Antibody for 4-1BB

ABSTRACT

The present invention includes the receptor protein 4-1BB and the cDNA gene encoding for receptor protein 4-1BB. The nucleotide sequence of the isolated cDNA is disclosed herein along with the deduced amino acid sequence. The 4-1BB protein and fragments and derivatives can be used: 1) as a probe to isolate ligands to receptor protein 4-1BB, 2) to stimulate proliferation of B-cell&#39;s expressing 4-1BB, or 3) to block 4-1BB ligand binding. A monoclonal antibody against 4-1BB was developed which specifically recognizes an epitope on the extracellular domain of receptor protein 4-1BB. The monoclonal antibody can be used enhance T-cell proliferation and activation by treating T-cells that have expressed receptor protein 4-1BB with the monoclonal antibody. The effectiveness of the treatment was enhanced when conducted in the presence of protein tyrosinase kinase. A fusion protein for detecting cell membrane ligands to receptor protein 4-1BB was developed. It comprises the extracellular portion of the receptor protein 4-1BB and a detection protein bound to the portion of the receptor protein 4-1BB.

[0001] This application is a continuation-in-part of co-pendingapplication Ser. No. 07/922,996, which is a continuation-in-part ofcopending application Ser. No. 07/267,577 filed Nov. 7, 1988.

[0002] The subject matter described herein was in part a subjectinvention of NIH Grants Nos. IR23AI23058-03, RO1 AI28175 and P60 KD20542of which the present inventor was the Principal Investigator and eitherthe Donald Guthrie Foundation for Medical Research Inc. of GuthrieSquare, Sayre, Pa. 18849-1669 or Indiana University School of Medicineof Indianapolis, Ind. 46202, was the Grantee.

FIELD OF THE PRESENT INVENTION

[0003] The present invention relates to a previously unknown receptorprotein which were isolated and identified based on specific expressionof the T cell genes using a technique identified by the present inventorin a publication (Proc. Natl. Acad. Sci. USA. 84, 2896-2900, May 1987,Immunology), and more particularly relates to the receptor protein,4-1BB, a monclonal antibody against 4-1BB, a ligand protein fordetecting the presence of 4-1BB binding sites on cells and methods ofusing these proteins and antibody.

BACKGROUND OF THE PRESENT INVENTION

[0004] Lymphokines are the proteins by which the immune cellscommunicate with each other. Scientists produce them in sufficientquantities for therapeutic use against immunologic diseases. The immunesystem of humans and other species requires that white blood cells bemade in the bone marrow, which white blood cells include phagocytes,lymphocytes and B cells. As presently understood, the phagocytes includemacrophage cells which scavenge unwanted materials such as virus proteinfrom the system. The lymphocytes include helper T cells and killer Tcells and B cells as well as other cells, including those categorized assuppressor T cells.

[0005] The B cells produce the antibodies. The killer T cells physicallypierce the cell and the helper T cells facilitate the whole process. Inany event, the immune process is facilitated by lymphokines. Interleukin1, secreted from macrophages activate the helper T cells and raise thebody temperature causing fever which enhances the activity of the immunecells. The activated helper T Cells produce Interleukin 2 andInterleukin stimulates the helper and killer T cells to grow and divide.The helper T cells also produce another lymphokine, B cell growth factor(BCGF), which causes B cells to multiply. As the number of B cellsincreases, the helper T cells produce another lymphokine known as the Bcell differentiating factor (BCDF), which instructs some of the B cellsto stop replicating and start producing antibodies. T cells also producea lymphokine, gamma interferon (IF), which has multiple effects likeInterleukin 2. Interferon helps activate killer T cells, enabling themto attack the invading organisms. Like BCGF, interferon increases theability of the B cells to produce antibodies. Interferon also affectsthe macrophages to keep them at the site of the infection and help themacrophages to digest the cells they have engulfed. Gathering momentumwith each kind of lymphokine signal between the macrophages and the Tcells, the lymphokines amplify the immune system response and the virusprotein or other foreign matter on the infected cells is overwhelmed.There are many other lymphokines, maybe a hundred or more, whichparticipate in the immune process. Many lymphokines are known and manyare not.

[0006] Lymphokines are sometimes called intercellar peptide signals.Among scientists there is widespread use of cloned cell lines aslymphokine producers and the isolation of lymphokine mRNA has become acommon technique. The present invention relates to a previously unknownreceptor protein which was isolated and identified based on specificexpression of the T cell genes using a technique identified by thepresent inventor in a publication (Proc. Natl. Acad. Sci. USA. 84,2896-2900, May 1987, Immunology). The protocol reported in thispublication can be used by scientists to detect virtually all of thelymphokines because the method is designed to detect virtually all themRNA expressed differentially and the mRNA sequences of the immune cellsare expressed differentially as they relate to the T cells and thekiller T cells even though the level of expression is low and thequantity of the secreted lymphokine protein is low. The present inventorbelieves that the analysis described in the above identified publicationcan reveal biologically important molecules such as lymphokines becausethere are many indications that biologically important or activemolecules are coded by the most scarce messages. An example is atransforming growth factor (TGF) which is present as only one of amillion clones. There are many known lymphokine proteins and theyinclude the interferons, interleukin-1,2,3,4,5,6,7, colony-stimulatingfactors, lymphotoxin, tumor necrosis factor and erythropoietin, as wellas others.

[0007] Most T cell factors have been classically identified byrecognizing biologic activities in assays, purifying the proteininformation. An alternative approach is to isolate putative T cell genesbased upon specific expression and then demonstrate the function of theunknown molecule. Using the aforesaid modified differential screeningprocedure, the present inventor has recently cloned a series of T cellsubset-specific cDNAs from cloned helper T (HTL) L2 and cloned cytolyticT lymphocyte (CTL) L3.

[0008] A series of T-cell subset-specific cDNAs were isolated fromcloned murine T-cells by employing a modified differential screeningprocedure (88, 89). The nucleotide sequence and expression properties ofsome of the cDNA species have been reported (90). One of the genes notpreviously characterized, 4-1BB, was studied further. Apparent fulllength cDNAs corresponding to fourteen species of the 16 initialisolates were sequenced and were found to constitute five differentspecies. Three of the five were identical to previously reported cDNAsequences of proenkephalin, T cell replacing factor and HF gene (aserine esterase). The other two, represented as L2G25B and 4-1BB, werenovel sequences of unknown function. The open reading frames of 4-1BBand L2G25B code for 245 and 92 amino acids, respectively. The predictedproteins of 4-1BB and L2G25B include 22 and 23 amino acid-long putativesignal sequences, respectively. The protein backbones of mature proteinsencoded by 4-1BB and L2G25B are composed of 234 amino acids withmolecular weight of 25000 and 69 amino acids with molecular weight of7880, respectively. 4-1BB contains two potential N-glycosylation siteswhile L2G25B has none. 4-1BB contains 23 cysteine residues in theputative mature protein.

[0009] The cDNA L2G25B encodes for the lymphokine, macrophageinflammatory protein-1α or MIP-1α. MIP-1α has been described in a paperentitled, “Enhancing and Suppressing Effects of Recombinant MurineMacrophage Inflammatory Proteins on Colony Formation In Vitro by BoneMarrow Myeloid Progenitor Cells”, Hal E. Broxmeyer, Barbara Sherry, LiLu, Scott Cooper, Kwi-Ok Oh, Patricia Tekamp-Olson, Byoung S. Kwon, andAnthony Cerami, Blood 76, 111-1116, 1990 and is incorporated herein byreference. This was the first time the suppressing activity of MIP-1αwas characterized.

[0010] The cDNA clone, called 4-1BB, was originally believed to be alymphokine based upon the early experiments disclosed herein. The laterstudies showed that 4-1BB is an inducible receptor-like sequence foundin both cytolytic and helper T-cells. Chalupny and colleagues (132)published a paper disclosing a fusion protein consisting of theextracellular domain of 4-1BB and the Fc region of IgG₁. Chalupny et al.taught that the highest levels of 4-1BB Rg (4-1BB-immunoglobulin fusionprotein) binding was to human vitronectin. The present inventorperformed an ELISA study using 4-1BB-AP (the fusion protein of thepresent invention) and human vitronectic. No binding of 4-1BB-AP basedon alkaline phoshatase activity was observed. To rule out thepossibility that 4-1BB-AP was binding to proteins extrinsically attachedto the cell surface, B-Cell lymphomas were washed in acid conditionsprior to the binding assay; 4-1BB-AP still bound specifically to matureB-cell lymphomas. Based on the data reported by Chalupny et al. it doesnot appear that they teach a fusion protein capable of accuratelyidentifying 4-1BB ligands.

SUMMARY OF THE PRESENT INVENTION

[0011] The present invention includes the receptor protein 4-1BB and thecDNA gene encoding for receptor protein 4-1BB. the nucleotide sequenceof the isolated cDNA is disclosed herein along with the deduced aminoacid sequence. The cDNA gene dentified as p4-1BB was deposited at theAmerican Type Culture Collection at 12301 Parklawn Drive, Rockville, Md.20852 under ATCC No. 67825. The cDNA, and fragments and derivativesthereof, can be used as a probe to isolate DNA sequences encoding forproteins similar to the receptor protein encoded- by the cDNA. Namely,the cDNA of a human receptor corresponding to the mouse cDNA 4-1BB canbe isolated from a human source using cDNA 4-1BB as a probe.

[0012] The receptor protein 4-1BB can be produced by: 1) inserting thecDNA of 4-1BB into an appropriate expression vector, 2) transfecting theexpression vector into an appropriate transfection host, c) growing thetransfected hosts in appropriate culture media and d) purifying thereceptor protein from the culture media. The protein and fragments andderivatives can be used: 1) as a probe to isolate ligands to receptorprotein 4-1BB, 2) to stimulate proliferation of B-cell's expressing4-1BB ligands, or 3) to block 4-1BB ligand binding. B-cells that haveexpressed a ligand to receptor protein 4-1BB are treated with cells thathave expressed receptor protein 4-1BB and B-cell proliferation isinduced. The use of 4-1BB to block 4-1BB ligand binding has practicalapplication in the suppression of the immune system during organtransplantation. A similar costimulatory immune system pathway is beinganalyzed for this type of application. See “Mounting a Targeted Strikeon Unwanted Immune Responses”, Jon Cohen, Science, Vol. 257, 8-7-92;“Long Term Survival of Xenogeneic Pancreatic Islet Grafts Induced byCTLA4Ig”, Lenschow et al, Science Vol. 257, Jul. 8, 1992; and“Immunosuppresion in Vivo by a Soluble Form of the CTLA-4 T CellActivation Molecule”, Linsley et al, Science Vol. 257 Jul. 8, 1992.

[0013] A monoclonal antibody against 4-1BB was developed whichspecifically recognizes an epitope on the extracellular domain ofreceptor protein 4-1BB. The monoclonal antibody is produced from ahybridoma identified as 53A2 and deposited at the American Type CultureCollection at 12301 Parklawn Drive, Rockville, Md. 20852 under ATCC No.:HB-11248. The monoclonal antibody can be used enhance T-cellproliferation by treating T-cells that have expressed receptor protein4-1BB with antiCD3 monoclonal antibody.

[0014] Some tumors are potentially immunogenic but do not stimulate aneffective anti-immune response in vivo. Tumors may be capable ofdelivering antigen-specific signals to T cells, but may not deliver theco-stimulatory signals necessary for full activation of T cells.Expression of the co-stimulatory ligand on B7 of melanoma cells wasfound to induce the rejection of a murine melanoma in vivo. (“TumorRejection After Direct Co-Stimulation of CD8⁺ T Cells by B7-TransfectedMelanoma Cells”, Sarah E. Townsend and James P. Allison, Science Vol.259, 1-5-93.) The monoclonal antibody of the present invention may becapable of the same effect as it is now know to enduce T cellproliferation and activation.

[0015] A fusion protein for detecting cell membrane ligands to receptorprotein 4-1BB was developed. It comprises the extracellular portion ofthe receptor protein 4-1BB and a detection protein bound to the portionof the receptor protein 4-1BB. The portion of the receptor protein 4-1BBbinds to the cell membrane ligands and binding can be detected byrelative activity assays for the detection protein. The fusion proteinis placed in the presence of a cell suspected to express the receptorprotein 4-1BB. Then the cell is washed of any fusion protein not boundto the cell membrane ligands. Once the washed cells are placed in thepresence of a substrate for the detection protein and the relativeactivity of the detection protein can be measured. The detection proteindisclosed herein is alkaline phosphatase.

[0016] The primary object is to provide the teachings identifying thenew receptor, 4-1BB as identified herein by its sequence.

[0017] Another object of the present invention is to provide teachingsof how the new receptor may be used to isolate and identifycorresponding molecules in related species.

[0018] Still another object of the teachings of the present invention isto teach the identification of the new receptor as reported herein.

[0019] Still another object of the teachings of the present invention isto teach the anti-4-1BB monoclonal antibody produced from the hybridoma53A2 as reported herein.

[0020] Still another object of the teachings of the present invention isto teach a fusion protein comprising the extracellular portion of 4-1BBand a detection protein.

[0021] Still another object of the teachings of the present invention isto teach methods of using the cDNA 4-1BB, the receptor protein 4-1BB,the monoclonal antibody and the fusion protein.

BRIEF DESCRIPTIONS OF THE FIGURES

[0022]FIGS. 1a and 1 b are flow sheets of the present inventor'sapproach to identifying L2 (helper T lymphocyte) specific and L3(Cytolytic T lymphocyte) specific cDNA clones.

[0023]FIGS. 2a and 2 b show the nucleotide sequence and the deducedamino acid sequence of mouse receptor 4-1BB.

[0024]FIGS. 3a, 3 b, and 3 c show an RNA blot analysis ofConA-stimulated L3 RNA with the expression being for different sizes ofreceptor 4-1BB mRNA.

[0025]FIG. 4 shows a Southern Blot analysis of mouse genomic DNA forfragments of L2G25B and 4-1BB cDNA.

[0026]FIGS. 5a and 5 b show L2G25B and 4-1BB expressed preferentially inL2 and L3 cells only after concanavalin A stimulation.

[0027]FIGS. 6a, 6 b, 6 c and 6 d show RNA Blot patterns of lymphokineL2G25B and receptor 4-1BB in mRNA expression TCR stimulation or Il-2treatment.

[0028]FIGS. 7a, 7 b and 7 c show expression of lymphokine L2G25B mRNAand receptor 4-1BB mRNA in a HTL L2 and a CTL dB45 cells.

[0029]FIGS. 8a and 8 b show the expression of receptor 4-1BB mRNA inconcanavalin A-stimulated hybridomas PN37 and Md90 and in a unstimulatedCTL CTLLA11.

[0030]FIGS. 9a, 9 b, 9 c and 9 d show the effect of cyclosporin A onL2G25B and 4-1BB mRNA expression.

[0031]FIGS. 10a, 10 b and 10 c shows the expression of lymphokine L2G25Band receptor 4-1BB mRNA in mouse splenocytes.

[0032]FIG. 11 shows the expression of 4-1BB on RNA in CTLL-R8.

[0033]FIG. 12 shows an immunoblot analysis of CTLL-R8 cell lysates withanti-4-1BB-0 serum.

[0034]FIG. 13 shows an immunoblot analysis of the 4-1BBPs.

[0035]FIG. 14 shows representative histograms of IgG fraction ofanti-4-1BB-O related fluorescence intensity of CTLL-R8 cells.

[0036]FIG. 15 shows the expression of 4-1BB RNA in mouse tissues.

[0037]FIG. 16 shows the histology of NOD mouse pancreata andimmunofluorescent staining of islets showing different stages ofinsulitis.

[0038]FIG. 17 shows a comparison of the 4-1BBP amino acid sequence withthe amino acid sequence in sina of Dorsophila and DG17 of Dictyostelium.

[0039]FIG. 18 shows a northern blot analysis of kidney and brain RNA.

[0040]FIG. 19 shows 4-1BB immunostaining in the cortex (a, b, and d)striatum (a, d, and e) at progressively enlarged magnifications.

[0041]FIG. 20 shows distinct 4-1BB immunopositive reaction in thecerebellum at three progressively enlarged magnifications (a, b, and c).

[0042]FIG. 21 shows purified re-4-1BBP in three bands of 18, 20, and 23,kDa (arrows): lane 1, Coomassie staining; lane 2, anti-4-1BB-O antibodystaining; lane 3, molecular size marker.

[0043]FIG. 22 shows recombinant 4-1BB protein fractionated on a 10% SDSpolyacrylamide gel.

[0044]FIG. 23 shows an immunoprecipitation of cell surface 4-1BB proteinsynthesized by T lymphocytes.

[0045]FIGS. 24a and 24B show that 4-1BB mRNA expression in inducedmurine splenic T cells by anti-CD3-activation.

[0046]FIG. 25 shows that optimal induction of 4-1BB mRNA requires bothProtein Kinase C activation and increases in intracellular Ca²⁺;thymocyte culture were stimulated with medium alone (lane 1), 1 μMionomycin (1) (lane 2), 10 ng/ml TPA (T) (lane 3), T+I (lane 4) for 6hours and monitored for 4-1BB mRNA expression by Northern analysis.

[0047]FIG. 26 shows a Northern blot analysis of Thymocytes cultures thatwere stimulated with medium alone (lane 1), 20 μg/ml CHX (lane 2), T+1(lane 3), or CHX+T+1 (lane 4) for 6 hours and monitored for 4-1BB mRNAexpression.

[0048]FIGS. 27a-d show that 4-1BB is expressed on the cell surface ofactivated thymocytes, splenic T cells, CD4⁺ and CD8⁺ T cells.

[0049]FIG. 28 shows a bar graph demonstrating that anti-4-1BB mAb, 53A2,enhances the prolifeation of anti-CD3-activated splenic T cells.

[0050]FIGS. 29a-c show specificity of 53A2 mAb to native and recombinant4-1BB.

[0051]FIGS. 30a and 30 b show the identification of thecoimmunoprecipitated proteins.

[0052]FIGS. 31a-c show an analysis of the association of 4-1BB andp56^(lck) in a baculoviral expression system.

[0053]FIGS. 32a-c show an analysis of the association of 4-1BB andp56^(lck) HeLa cells.

[0054]FIG. 33 is a Western analysis that shows the expression of the4-1BB-AP fusion protein and rs4-1BB.

[0055] FIGS. 34 a-c show the quantitative analysis of 4-1BB-AP bindingto lymphoid- and nonlymphoid-cell lines.

[0056]FIGS. 35a and 35 b show the characterization of 4-1BB-AP bindingto A20 B-cell lymphoma cells.

[0057]FIG. 36 shows the costimulation of anti-r-primed B cells withfixed-SF21-4-1BB cells.

DETAILED DESCRIPTION

[0058] In the following detailed description a successive series ofstudies are presented which characterize the receptor 4-1BB. Referencesare made to known procedures and studies, as well as published work ofthe applicant. These publications are incorported herein by referencefor clarity and listed in an appendix included at the end of thisdetailed description.

[0059] The following abbreviations are used herein: CTL, cytolytic Tlymphocyte; HTL, helper T lymphocyte; LGL, large granular lymphocytes;NK, natural killer cells; SDS, sodium dodecysulfate; SSC, 150 mM sodiumchloride/15 mM sodium citrate, pH 7.0; TPA,12-O-tetradecanoylphorbol-13-acetate. Th, helper T lymphocytes; IL-2,interleukin 2; IL-3, interleukin 3; rIL-2, recombinant Il-2; CSF-GM,granulocyte/macrophage colony-stimulating factors; cRNA, complementaryRNA; ss, single-stranded; ds, double-stranded; TCR, T-cell antigenreceptor; PTA, phorbol 12-tetradecanoate 13-acetate; r, recombinant; mu,murine; hu, human; MIP, macrophage inflammatory protein; BFU-E, burstforming unit-erythroid, an erythroid progenitor cell; CFU-GEMM, colonyforming unit-granulocyte erythroid macrophage, megakaryocyte, amultipotential progenitor cell; CFU-GM, colony forming unit-granulocytemacrophage, a granulocyte-macrophage progenitor cell; CFU-S, colonyforming unit-Spleen, a multipotential stem cell; H-ferritin, the heavychain subunit form of ferritin; MGF, mast cell growth factor, a c-kitligand; CSF, colony stimulating factors; G, granulocyte; M, macrophage;Epo, erythropoietin; IL, interleukin; LD, low density; NALDT⁻,non-adherent low density T-lymphocyte depleted; PMSF,phenylmethylsulfonyl fluoride; PBS, phosphate buffered saline; MIP-1α-R,MIP-1α receptor; rMIP-1α, recombinant MIP-1α protein; nMIP-1, nativemacrophage inflammatory protein-1; AcNPV, Autographa californica nuclearpolyhedrosis virus; SDS, sodium dodecyl sulfate; LPS,lipopolysaccharide; ConA, concanavalin A; DTT, dithiothreitol; mAb,monoclonal antibody.

[0060] Initial Isolation and Sequencing of 4-1BB

[0061] Materials and Methods

[0062] Cells cloned murine CTL L3 cells (1), are thy-1,2⁺, Lyt-2⁺,LFA-1⁺, LeT4⁻ and H-2L^(d) reactive. Cloned murine HTL L2 cells (2) areThy-1,2⁺, LFA-1⁺, Lyt-2⁻, LeT4⁺ and M1s^(a/d) reactive.

[0063] Methods of isolating and maintaining the cloned helper Tlymphocytes (Th), L2, and the cloned cytolytic T lymphocytes (CTL), L3,have been described in the above identified publication. To stimulatethe cloned T cells, we resuspended them at 10⁶-10⁷ cells per ml andcultured them with Con A (Pharmacia) at 10 ug/ml for L2 cells or 2 ug/mlfor L3 cells or human recombinant IL-2 (rIL-2; Cetus) at 102-103units/ml. Immobilized clonotypic monoclonal antibody 384.5, which reactswith the TCR of L3 cells (2), was used to stimulate L3 cells.

[0064] Mouse thymoma cells, EL4, and mouse B-cell lines, A20.2j and K46,were maintained in RPMI 1640 medium containing 5% fetal calf serum. EL4cells were stimulated with phorbol 12-tetradecanoate 13-acetate (PTA; 10ng/ml) for up to 20 hr, monitoring the stimulation by IL-2 assay (3).

[0065] cDNA Libraries. RNAs of L2 and L3 cells that were stimulated byCon A for 14 hr, were extracted (4) and poly(A)⁺ mRNA was purified on anoligo(dT)-cellulose column (5). Double-stranded (ds) cDNA wassynthesized from the poly(A)⁺ mRNA (6). The cDNA was methylated at EcoRIsites, EcoRI linkers were ligated to cDNA, and then the cDNA wasenriched for molecules larger that 250,000 daltons by passage overBio-Gel A-150m columns. The cDNAs were inserted into the EcoRI Site ofgt10 bacteriophage cloning vector (7).

[0066] cDNA Probe. Six micrograms of poly(A) mRNA was denatured with 10mM methylmercuric hydroxide and incubated in a buffer containing 100 mMTris HCI at pH 8.3, 50 mM KCl, actinomycin D at 50 mg/ml, 30 mM2-mercaptoethanol, 10, mM MgCl₂, (dt₁₂₋₁₈ at 5 ug/ ml, 0.5 mM each ofdATP, dCTP, and dGTP, 0.01 mM dTTP, 0.001 mM [α-³²P]dTTP (3000 Cimmol⁻¹; 1 Ci=37 GBq), and reverse transcriptase from avianmyeloblastosis virus at 1000 units/ml at 46° C. for 30 min.Single-stranded (ss) cDNA was freed from its template RNA by incubationin 200 mM NaOH/10 mM EDTA at 60° C. for 30 min and passed over a 4-mlcolumn of Sephadex G-100. The specific activity of the probe was usually1.6-2.0×10⁸ cpm/ug of cDNA.

[0067] Subtracted cDNA Probe. The ss cDNA prepared from L2 RNA washybridized to a R_(o)t of 1200-1500 (mol of nucleotide per liter)×secwith poly(A)⁺ mRNA of A20.2j in 0.41 M sodium phosphate buffer, pH 6.8,containing 0.1% NaDod-SO₄ and 1 mM EDTA, in a volume of 25-50 ul. The sscDNA fraction was collected by chromatography through a hydroxylapatitecolumn as recommended by the vendor (Bio-Ran). Seven percent of inputcDNA was recovered in the ss fraction and used for a second round ofhybridization to A20.2j poly(A)⁺ mRNA to an equivalent R_(o)t of 500(mol/liter)×sec. Approximately 93% of initial input radioactivity wasrecovered. Starting with 6 ug of poly(A)⁺ mRNA, approximately 5.5×10⁶cpm was obtained as a probe.

[0068] DNA and RNA Blot Hybridization. Recombinant phage DNA wasprepared (8) and digested with EcoRI. DNA fragments were transferred toGeneScreenPlus membranes (New England Nuclear) and hybridized with sscDNA probes (9). RNA was run on 1.2% formaldehyde denaturing agarose gel(10) and transferred to GeneScreenPlus. Probes for RNA hybridizationwere prepared from gel-purified cDNA inserts by the random primingmethod (11). Total cytoplasmic RNA of poly(A)⁺ RNA were fractionated on1.2% agarose-formaldehyde gels and transferred to GeneScreenplus (NEN,Boston, Mass.). Gel-purified cDNA inserts were [32p] labeled by nicktranslation and used as probes. When a Northern blot of GeneScreenpluswas used multiple times for hybridization, the previous probe wasremoved by treating the membrane in 10 mM Tris-Hcl (pH 7.0) and 0.2% SDSat 85° C. for 1 hr.

[0069] High molecular weight DNA of mouse spleens was prepared asdescribed previously (12). Endonuclease digests of DNA wereelectrophoresed in 0.8T agarose gel at 4° C. The DNA was denatured, andtransferred to GeneScreenPlus as described by Southern (13). The blothybridized with [³²P]-labeled cDNA inserts. L2 cells were stimulatedwith concanavalin A (10 ug/ml) for 14 hr, at a cell concentration of10⁶-10⁷/ml. L3 cells were stimulated with concanavalin A (2 ug/ml) for14 hr, at a cell concentration of 2.5×10⁶/ml. Mouse thymoma EL-4 cells(14) were stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA, 10ng/ml) at a cell concentration of 1.0×10⁶/ml for 20 hr; stimulation wasmonitored by Il-2 assay (3). B cell lymphoma K46 (15), and rat Nk cellLGL (16) were not stimulated with any of above reagents.

[0070]FIGS. 1a and 1 b are flow sheets of the present inventor'sapproach to identify L2-Specific and L3-specific cDNA clones.Therein: 1) the * means that preparation of subtracted L2 cDNA probe isdescribed in Materials and Methods; 2) the +means the probes of knownsequences were prepared for cDNAs of granulocyte/macrophagecolony-stimulating factors (CSF-GM), interleukin 3 (IL-3), IL-2, TCRα-chain, TCR β-chain, c-myc, and c-fos; 3)++ means the insert of eachnegative recombinant phage DNA was gel-purified and used as a probe forRNA blot hybridization (“Northern”) analysis of K46, unstimulated orPTA-stimulated EL4, large granular lymphocytes (LGL), and unstimulatedor Con A-stimulated L2 and L3. The [³²P]-labeled cDNA probe, preparedfrom poly(A)⁺ mRNA of A20.2j, was used to screen the library. The totalcDNA probe could detect a clone corresponding to 0.02-0.05% of the testmRNA (17). Of 18,000 plaques from the L2 cDNA library, 614 (3.4%) failedto hybridize to the B-cell cDNA probe. The subtracted L2 cDNA probe washybridized to these 614 plaques and 114 (18%) gave a signal; 372 plaquesgave no signal to the subtracted L2 cDNA probe or B-cell cDNA probe. Ofthose 372, 72 clones (19%) contained cDNA inserts. The 186 (114+72)clones from the L2 cDNA library were subjected to further analysis.

[0071] By similar analysis of approximately 8000 L3 cDNA clones, 150plaques ( 2.0%) that failed to hybridize to P-labeled B-cell cDNA probewere selected. Instead of screening the 150 plaques with subtracted L2cDNA probe, we digested recombinant phage DNA of each clone with EcoRIand immobilized the fragments on the filter. ³⁵S-labeled B-cell cDNAprobe was used to hybridize to the filters. The use of 35S for cDNAlabeling and Southern analysis increased the sensitivity at least5-fold. Fifty-six inserts (FIG. 1b) from L3 were identified, each ofwhich failed to hybridize to the B-cell cDNA probe.

[0072] One Hundred and eighty-six L2 cDNA inserts and 56 L3 cDNA insertswere hybridized to cDNAs of CSF-GM, IL-3, IL-2, TCR α-chain, TCRβ-chain, c-myc, and c-fos. Twelve clones hybridized to cDNA, for IL-3, 6to CSF-GM, 3 to IL-2, 2 to TCR β-chain, and 1 each to TCR α-chain andc-myc (Table 1). Twenty-nine clones whose cDNA inserts were less than 50base pairs (bp) were eliminated from further study. The blots containing132 L2 cDNA and 54 L3 cDNA inserts were hybridized to S-labeled ss cDNAprobe prepared from poly(A)⁺ mRNA of unstimulated L3 or of unstimulatedL2, respectively. Sixty-one inserts of L2 cDNA hybridized to the L3 cDNAprobe and 14 inserts of L3 cDNA hybridized to the L2 cDNA probe. TABLE 1T-cell specific cDNA clones isolated from L2 and L3 cDNA library Numberof Origin Group cDNA clone times isolated L2  1 CSF-GM     6  2 IL-3*   12  3 IL-2     3  4 TCR α-chain     1  5 TCR β-chain     2  6 c-myc    1  7 pBK791     4  8 pBK642     1  9 pBK671     1 10 pBK631     3 11L2G53#3     1 12 L2G95#3     1 13 L2G95#4     1 14 L2G25#4     1 15L2S35#3     1 Total 39 L3  1 TCR β-chain     2  2 L3G29#4     1  3L3G25#4     1  4 L3G14#2     1  5 L3G10#6     1  6 L3G7#1     1  7L3G18#3     1  8 L3G26#1     2 Total 10

[0073] T-cell-specific cDNA clones were isolated from ˜18,000 clones ofL2 library and 8000 clones of L3 library. After enrichment ofT-cell-specific sequences, cDNA clones for CSF-GM, IL-3, IL-2, TCRα-chain, TCR β-chain, and c-myc were detected by hybridization with thecorresponding full-length cDNA provided by other laboratories. Bycross-hybridization, the other clones (14 from L2 and 8 from L3) turnedout to represent 16 different genes (9 from L2 and 7 from L3). ThosecDNA clones representing 16 different genes were subjected to furtheranalysis.

[0074] A partial sequence analysis revealed that the IL-3-related clonescontained two different species.

[0075] The 71 (132-61) inserts from L2 and 40 (54-14) inserts from L3were used as probes with blots of 10 ug of poly(A)⁺ mRNA from K46, LGL(rat NK cell) (18), unstimulated or PTA-stimulated EL4, and 10 ug oftotal RNA from unstimulated or Con A-stimulated L2 or L3 cells.

[0076] Among these inserts, 29 (˜40%, 29/71) from L2 and 19 (˜47%,19/40) from L3 hybridized to K46 or all lanes. Fourteen inserts (˜20%,14/71) from L2 hybridized only to Con A-stimulated L2, or both of L2 andL3 RNA. Those cDNA inserts represented nine different cDNAs. From L3, 8(20%, 8/40) were T-cell specific, representing seven different genes;one gene was inducible by Con A in both L2 and L3, three genes wereexpressed constitutively and inducible by Con A only in L3 cells; andthe rest were inducible by Con A in L3 cells.

[0077] ***

[0078] A in L3 cells but not found in unstimulated L3 cells.Twenty-eight inserts ( 40%, 28,71) from the L2 cDNA library and 13inserts (-32%, 13/40) from the L3 library did not hybridize to any ofthe RNAs. Because less L2 or L3 RNA was available for blot hybridizationanalysis, we have not been able to eliminate the possibility that thoseinserts not expressed in K46, EL4, or LGL could still be expressed in L2or L3 at a low level.

[0079] Screening of cDNA Library and DNA Sequencing. L2 and L3 cDNAlibraries which were previously prepared were rescreened with cDNAinsert of each of 14 T Cell-specific genes. Typically 10 positive cloneswere chosen for each species and the sizes of cDNA inserts weredetermined. The longest cDNA inserts were employed for nucleotidesequence analysis. DNA restriction fragments, subcloned in M13 vectors(19), were sequenced by the dideoxy chain termination technique (20)employing Sequinase (U.S. Biochemical, Cleveland, Ohio), withmodification made to accommodate 2″-deoxyadenosine 5″-[α[³⁵S]thio]triphosphate (21).

[0080] Nucleotide and Protein Sequence Comparison. Full length cDNA andpredicted protein sequence were compared with the sequences in theGeneBank (NIH) DNA Sequence Library, European Molecular BiologyLaboratories (EMBL) and National Biomedical Research Foundation (NBFR).Predicted protein were analyzed by Pepplot program.

[0081] Table 2 summarizes T cell cDNAs identified from 14 hrConA-stimulated L2 and L3 cDNA libraries. Besides the cDNAs listed inthe table, CSF-GM, IL-2, IL-3, α-, β-T cell receptor and c-myc cDNAswere identified by cross-hybridization of T cell enriched cDNAs with thecorresponding full-length cDNA provided by other laboratories. TABLE 2SUMMARY OF cDNA CLONES IDENTIFIED Full cDNA Clone* Length IsolatedSpecificity of cDNA Previously Expression Identification 4-1BB L3G29$3,L2 and L3 previously unknown L3G25#4, L3G14#2 L2G25B L2G25#4, L2 and L3previously unknown L2G95#4, (MIP-1α) L2G53#3, (related to PLD78) L2G95#3L2S35 L2S35#3, L2 only proenkephalin L2PBK671 8-1R L2PBK79J, L2 and EL-4T cell replacing L2PBK642, factor L2PBK631 L3G10 L3G10#6, L3 only HFgene L3G18#3 (serine esterase) N.D.** L3G7#1 L3 AND EL-4 unknown N.D.**L3G26#1 L3 and EL-4 unknown

[0082] Among the 16 unidentified T cell genes two representedproenkephalin which was identical to the sequence reported by Zurawskiet al (22), three were T cell replacing factor (23), and two representedT cell serine esterase gene (24).

[0083] Four species were from different regions of cDNA represented asL2G25B( 800 bases pairs). L2G25B was homologous to a human cDNA PLD 78915) of unknown function. Three Species (L3G29#4, L3G25#4 AND L3G14#2)were from different regions of 4-1BB (2,400 base pairs). There were noreports of sequences homologous to 4-1BB. L3G7#1 and L3G26#1 were notcharacterized vigorously since we could not isolate longer inserts andtheir expression was very low in L3.

[0084] In the previous studies, 13 L3 cDNAs were isolated whosespecificity were not assigned by RNA blot analysis. One of them 03-1)was 64% homologous to reported T cell serine esterase (25). The sequencewas reported as a new member of T cell serine esterase (26).

[0085] The nucleotide sequence of three overlapping cDNA clonesrepresented by 4-1BB was determined according to the strategy shown inFIG. 2a. The nucleotide sequence of 4-1BB revealed a single long openreading frame, beginning with the ATG codon at nucleotide residues 1-3(FIG. 2b.). This reading frame codes for a polypeptide of 256 aminoacids with a molecular weight of 27,587. The assigned ATG is preceded byan in-frame termination codon TGA (nucleotide residues −12 to 9). Thesequence flanking the assigned ATG (nucleotide residues −5 to 4) is afavored sequence for eukaryotic initiation sites (consensus;CCG/ACCATGG) described by Kozak (30). In fact, 8 out of 9 consensussequences were identical to the sequences flanking to the assignedinitiation codon. The codon specifying carboxy-terminal leucine isfollowed by the translational termination codon TGA (nucleotide residues659-771). 4-1BB contains 1434 nucleotides of 3′-untranslated regionwhich did not extend as far as polyadenylation signal nor the poly (A)⁺tail.

[0086]FIG. 2 shows the nucleotide sequence and the deduced amino acidsequence of 4-1BB. The nucleotides of the message strand are numbered inthe 5′ to 3′ direction and numbers are shown on both sides of thesequence. Nucleotide residue 1 is the A of the initiation codon ATG, andthe nucleotides on the 5′ side of residue 1 are indicated by negativenumbers. The predicted amino acid sequence is shown below the nucleotidesequence. Putative signal peptide is underlined. The potentialasparagine-linked glycosylation sites are underlined. Potentialpolyadenylation signal is boxed. Stop codon is indicated by (---).Cysteine residues are highlighted by (•). An unusual feature of 4-1BBsequence is that there is a potential polyadenylation signal of AATAAAat nucleotides 1158-1163 (FIG. 2b boxed). It was believed that thissignal was functional because this gene produces at least two differentsizes of mRNA. We believe that this signal of AATAAA at nucleotides1158-1163 (FIG. 2b boxed). We believe that this signal is functionalbecause this gene produces at least two different sizes of mRNA. FIG. 3aand b shows RNS blot analysis of ConA-stimulated L3 RNA. When the blotwas hybridized to L3G25#4 probe which contained sequences of 3′ side tothe polyadenylation signal (nucleotides 1284-1557). The probe detectedone RNA species of approximately 2.4 kb. When the same blot washybridized to L3G14#2 probe which contained sequences of 5′ side to thefirst polyadenylation signal (nucleotides 661-855), the probe detectedtwo mRNA species of approximately 1.5 kb and 2.4 kb.

[0087]FIG. 3 shows the expression of two different sizes of 4-1BB mRNA.Ten micrograms of poly(A)⁺ mRNA from mouse B cell line (K46),TPA-stimulated EL-4 (EL-4 TPA) and rat NK cell line (LGL), and tenmicrograms of total RNA from unstimulated L3 (L3) and concanavalinA-stimulated L3 (L3 ConA) were fractionated on a 1.4% formaldehydeagarose gel, transferred to GeneScreenPlus and hybridized to[³²P]-labeled L3G25#4 (a), L3G14#2 (b) and L3G20#3 (c) sequentially.L3G25#4 and L3G14#2 represent cDNA fragments of the 3′ side and %′ sideto boxed AATAAA sequence, respectively.

[0088] L3G20#3 is an anonymous cDNA from L3 cDNA library and is used toshow that each lane of the blot contains a similar amount of RNA.Positions of 28S and 18S rRNA markers are each indicated. Arrowsindicate the specific hybridization signal.

[0089] The deduced sequence of the first 22 amino acids of 4-1BB hascharacteristics of the signal peptide of secretory andmembrane-associated protein (27), which mainly contains hydrophobicamino acids. We putatively assigned the first 22 amino acids as a signalpeptide. A possible cleavage site of the signal peptide is after glycineresidue at alanine (FIG. 2b). Gly-ala at amino acid\positions 22 and 23is one of the favorable signal peptidase cleavage sites(*). Thus theprotein backbone of processed 4-1BB protein is composed of 234 aminoacids with a molecular weight of 25,000. We found two potentialasparagine-linked glycosylation signals (22,23) at amino acid positions129 and 138 as underlined in FIG. 2b. The predicted 4-1BB proteincontains unusually large numbers of cysteines. There are 23 cysteineresidues in the putative mature protein as dotted in FIG. 2a.

[0090] There is a stretch of 26 amino acids that constitutes hydrophobicdomain toward the carboxy terminus of the protein (amino acids atpositions 182-211). Whether this region serves as a membrane-spanningdomain is not known. This region is followed by the 45 amino acids whichconstitute a hydrophilic region.

[0091] Southern Blot Analysis. As shown in FIG. 4, fragments of L2G25Band 4-1BB cDNA each detect single restriction fragment of approximately15 kb and 18 kb in both C57BL/6 and BALB/c, DNA, respectively. The dataindicate that the genes encoding the two molecules exist as a singlecopy in C57BL/6 and BALB/c mice. FIG. 4 shows a Southern Blot analysisof mouse genomic DNA. Genomic DNA from C57BL/6 (lanes 1,3) and BALB/c(lanes 2,4) was digested with EcoRI restriction enzyme, fractionated ona 0.8% agarose gel, transferred to Gene-Screenplus and hybridized to[³²P] labelled L2G25B (lanes 1,2) and 4-1BB (lanes 3,4).

[0092] The protocol developed by the present inventor and reported andpublished as identified hereinabove for a modified differentialscreening of a cDNA library by which one can detect a broadrepresentation of the mRNA expressed differentially in two differentcell types, was applied to the systematic analysis of HTL and CTL geneexpression and allowed us to isolate T cell subset specific genes. Thisapproach offers an alternative to the classical protein purification foridentifying molecules and genes. Advantages of this method are: 1) Theapproach identifies the existence of molecules which otherwise may bedifficult or impossible to recognize or isolate; 2) Even molecules whichexist at a low level in the natural source can be produced in quantityby recombinant DNA technologies and in turn provide enough protein topermit study of function and possibly clinical applications; and 3) Itis a straightforward method for identifying mutations of the gene usingthe nucleic acid probe. As an illustration of the usefulness of thisapproach, the genes for T-cell antigen receptors and X-linkedimmunodeficiency (xid) genes were cloned and characterized in thisfashion (31). This approach has already proven to be useful in isolatingknown as well as previously unrecognized T-cell mediators.

[0093] Using the same concanavalin A stimulated L2 cells, Prystowsky etat (32) identified 10 different lymphokine activities from culturesupernatants; they include IL-2, IL-3 BCSF, CSF, IFN- and fiveunidentified factors which affect macrophage activities. In the courseof the studies cDNAs were isolated and identified for IL-2, IL-3, CSF, Tcell replacing factor and proenkephalins from the concanavalinA-stimulated L2 and cDNA library (2 and unpublished observations).Therefore, it was considered possible that L2625B and 4-1BB representedthe unidentified soluble mediators of Prystowsky et al which affectmacrophage activities.

[0094] By applying a modified differential screening of L2 and L3 cDNAlibrary, two novel T cell genes were isolated. Correlation of these Tcell molecules with functional activities was shown by the followingevidence. L2G25B was shown to code for a lymphokine and 4-1BB was shownto have similar activity, however, 4-1BB was later shown to be areceptor protein.

[0095] T-cell-specific expression of L2G25B and 4-1BB. L2G25B wasisolated from an L2 cDNA library, and 4-1BB was isolated from and L3cDNA library by the aforesaid modified differential screening (5). Asshown in FIGS. 6a and 6 b, L2G25B and 4-1BB were expressedpreferentially in L2 and L3 cells only after concanavalin A stimulation.The sizes of transcripts were approximately 800 bases for L2G25B and2400 bases for 4-1BB. The abundance of the two transcripts was 5-10 foldhigher in L2 cells than in L3 cells. The two transcripts were notdetectable in K46 B cells, EL-4 thymoma cells or rat large granularlymphocytes. L2G25B mRNA was consistently more abundant than 4-1BB mRNA.FIG. 5 shows T cell-specific expression of L2G25B and 4-1BB mRNA. Poly(A)⁺ mRNA was prepared from mouse B cell line (K46), unstimulated EL-4(EL-4), TPA-stimulate EL-4 (EL-4 TPA) and rat NK cell line (LGL), andtotal RNA was prepared from unstimulated L2 (L2), concanavalinA-stimulated L2 (L2 ConA), unstimulated L3 (L3) and concanavalinA-stimulated L3 (L3 ConA). Ten micrograms of total RNA or ten microgramsof poly(A)⁺ RNA was fractionated on a formaldehydelagarose gel,transferred to GeneScreenplus and hybridized to [P]-labelled L2G25B(a)and 4-1BB(b) sequentially. Positions of 28S and 18S rRNA markers areeach indicated. An arrow indicates the specific hybridization signal.

[0096] L2G25B and 4-1BB mRNA were inducible by TCR stimulation. but notby Il-2 stimulation. The inducibility of the two cDNA clones was testedafter L3 TCR stimulation by clonotypic antiTCR mAb, 384.5, or Il-2. Asshown in FIGS. 6a and 6 b, the expression of the two cDNA was inducibleby TCR stimulation but not by Il-2 stimulation in L3 Cells. L2625B mRNAwas detectable at 0.5 hr after TCR stimulation, peaked at 6 hr, anddecreased thereafter until at least 24 hr. 4-1BB mRNA was detectable ata very low level in unstimulated L3 cells in this experiment. Theinduction of 4-1BB mRNA occurred approximately 6 hr after TCRstimulation and remained level until 24 hr.

[0097]FIG. 6c shows the kinetics of IFN-δ mRNA expression in the sameRNA blot as used in FIGS. 6a and 6 b. IFN-δ mRNA was detectable at 0.5hr after TCR stimulation, peaked at 12 hr and declined slightly until 24hr. There was a low level of IFN-δ mRNA in unstimulated L3 cells. Whenwe compared the peak level of L2G25B and 4-1BB mRNA with that of IFN-δmRNA, IFN-δ mRNA was at least 20 fold higher than that of L2G25B mRNAand a least 50 fold higher than that of 4-1BB mRNA. FIG. 6d demonstratesthat all six lanes contained almost identical amounts of RNA. The probewas a serine protease cDNA (L3G10#6) isolated from L3 cells. In summary,the pattern of the two cDNA expression was similar to that of IFN-6expression. FIG. 6 shows patterns of L2G25B and 4-1BB mRNA expressionafter TCR stimulation or IL-2 treatment. L3 cells were stimulated withclonotypic antiTCR mAb 384.5 for 0, ½, 6, 12 or 24 hr or with rIl-2 for6 hr. Ten ug of total RNA was fractionated on a formaldehyde/agarosegel, transferred to GeneScreenplus and hybridized to [³²P]-labeledL2G25B(a) 4-1BB(b), IFN-δ (c) and L3G10#6(d) cDNA. L3G10#6 is a serineprotease cDNA isolated from L3 cell cDNA library, which was identical toHF gene (24). L3G10#6 was used to show that each lane contains almostequal amounts of RNA. Positions of 28S and 18S rRNA markers are eachindicated. An arrow indicates the specific hybridization signal.

[0098] L2G25B and 4-1BB mRNA are inducible by TCR stimulation in othercloned HTL. CTL and hybridomas. As shown in FIGS. 7a and 7 b, L2G25B and4-1BB mRNA are also inducible in HTL L2 and CTL dB45 after TCRstimulation with antiTCR mAb F23.1. The mRNA level for the two cDNA wasalso much lower than that of IFN-δ in L2 and dB45 cells (FIG. 7c). L2cells show the highest level of expression of the three cell clones.FIG. 7 shows expression of L2G25B and 4-1BB mRNA in HTL L2 and a CTLdB45 cells. HTL L2 and CTL dB45 cells were stimulated with antiTCR mAbF23.1 for 6 hr. L3 cells were stimulated with anti TCR mAb 384.5 for 6hr. Ten ug of total RNA from unstimulated L3 (lane a) and stimulated L3(lane 2), unstimulated dB45 (lane 3), stimulated dB45 (lane 4),unstimulated L2 (lane 5) and stimulated L2 (lane 6) was fractionated onformaldehyde/agarose denaturing gel, transferred to GeneScreenplus andhybridized to [³²P] labeled L2G25B(a), 4-1BB(b), and IFN-δ (c) cDNA. Afraction of RNA in each lane was degraded and detected as RNAs in lowermolecular sizes. 4-1BB mRNA was inducible by concanavalin A in twocytotoxic hybridomas, PN37 and Md90 (FIG. 8a) and detectable inunstimulated CTLLA11 CTL (FIG. 8b) clones.

[0099]FIG. 8 shows expression 4-1BB mRNA in concanavalin A-stimulatedhybridomas PN37 and Md90, and in an unstimulated CTL CTLLA11. FIG. 8ashows BW5147, PN37 and Md90 cells were stimulated with concanavalin Afor 4 hr. Ten ug of poly(A)⁺ mRNA from unstimulated and stimulated eachof these cells was fractionated, transferred to nitrocellulose filterand probed with [³²P]-labelled 4-1BB cDNA. FIG. 8b shows ten ug ofpoly(A)⁺ mRNA from mouse melanoma cells (melanocyte) and ten ug of totalRNA from unstimulated L2 (L2), CTLLA11 (A11) and L3 (L3) cells wasfractionated, transferred to Gene-Screenplus and hybridized to [32p]4-1BB cDNA.

[0100] Effects of cyclosporin A on L2G25B and 4-1BB transcription. Totest the possibility that the two cDNAs represent two different solubleextracellular mediators, we next examined the effect of cyclosporin A onRNA expression. Cyclosporin A inhibits mitogen or antigen-induced T-cellproliferation (33). It has also been shown to block the induction ofexpression of several lymphokine genes including Il-2 and IFN-δ ((34).The inhibition of lymphokine production occurs at a pretranslationallevel (35). In contrast cyclosporin A appears to have no effect on theinducible expression of c-fos and Il-2 receptor genes in T cells. Asshown in FIGS. 9a and 9 b, cyclosporin A inhibited the inducedaccumulation of L2G25B and 4-1BB mRNA. The same findings were seen withIFN-δ (FIG. 9c). A low level of expression of L2G25B mRNA was seen inTPA-stimulated E1-4 cells in this experiment. FIG. 8d shows thatcyclosporin A had minimal or no effect on the level of serine protease(probe, L3610#6) mRNA and shows that the three lanes contained almostequal amounts of RNA (EL-4 or K46 cells did not express L3G10#6 mRNA).This data strongly suggested that L2G25B and 4-1BB expression would showsome of the same activation requirements as other known lymphokines.

[0101]FIG. 9 shows the effect of cyclosporin A on L2G25B and 4-1BB mRNAexpression. L3 cells were stimulated with concanavalin A, concanavalinplus cyclosporin A or concanavalin IA plus actinomycin D. Ten ug oftotal RNA from unstimulated L3(L3), concanavalin A-stimulated L3 (L3ConA), concanavalin A plus cyclosporin A-treated L3 (L3 ConA+CsA) andconcanavalin A plus actinomycin D-treated L3 (L3 ConA+ActD) cells andten ug of poly(A) mRNA from K46 (K46) and TPA-stimulated EL-4 cells(EL-4) were fractionated, transferred to GeneScreenplus membrane andhybridized to [³²P] labelled L2G25B(a), 4-1BB(b), IFN-δ (c) andL3G10#6(d) cDNA. Cyclosporin A treatment did not alter the level ofL3G10#6 mRNA but almost completely abrogated the induced expression ofother 4 mRNA species. An arrow indicates a specific hybridizationsignal.

[0102] L2G25B and 4-1BB mRNA were inducible in normal mouse spleencells. To find out if the expression of these genes were not unique tocertain cloned T cells or hybridoma cells, splenocytes from C57BL/6, andBALB/c mice were stimulated with concanavalin A and tested for mRNAexpression. As shown in FIG. 10a and 10 b the two mRNA were detectableafter concanavalin A stimulation in C57BL/6 and BLAB/c mousesplenocytes. They were also inducible in Swiss Webster mouse splenocytes(data not shown). As shown in FIG. 9c IFN-δ mRNA was detectable inconcanavalin A-stimulated BALB/c splenocytes (for unknown reasons IFN-δmRNA was not detectable in concanavalin A-stimulated C57BL/6 splenocytesin this experiment). RNA preparations for Figure c were different fromthose for Figures a and b. This data suggested that these moleculescould be induced in normal mouse spleen cells by appropriate stimulii asin the cloned T cells.

[0103]FIG. 10 shows the expression of L2G25B and 4-1BB mRNA in mousesplenocytes. Splenocytes were obtained from C57BL/6 and BALB/c mice andstimulated with concanavalin A for 14 hr. Ten ug of total RNA fromunstimulated BALB/c (lane 1) and stimulated BALB/c (lane 2),unstimulated C57BL/6 (lane 3) and stimulated C57BL/6 (lane 4)splenocytes was fractionated, transferred to Gene Screen plus endhybridized to [³²P]-labelled L2G25B(a), 4-1BB(b) and IFN-δ (c) cDNA. Aportion of L3G29 cDNA (approximately 200 pairs in the middle of themolecule) consistently detected an additional RNA species ofapproximately 1500 bases. The additional hybridization signal is seen inFIG. 10b.

[0104] L2G25B and 4-1BB share properties which suggested that theyencode soluble T cell mediators. The properties are; 1) the mRNA of thetwo was preferentially expressed in T cells; 2) The mRNAs of the twogenes were present in undetectable amount in T cells until induced byconcanavalin A, or by TCR stimulation; 3) The small size of the mRNA ofL2G25 was consistent with features of several analyzed lymphokine cDNAssuch as interleukins 2,3 and 5; 4) The patterns of expression were verysimilar to that of the lymphokine IFN-δ; 5) Both had a potential signalsequence and an AT rich 3′ untranslated region consistent with alymphokine gene (28) and 6) Cyclosporin A inhibited the induced mRNAexpression corresponding to the two cDNAs. Using the same concanavalin Astimulated L2 cells, Prystowsky et al (32) identified 10 differentlymphokine activities from culture supernatants; they included IL-2,IL-3, BCSF, CSF, T cell replacing factor and proenkephalins from ourconcanavalin A-stimulated L2 cDNA library (5 and unpublishedobservation). Therefore, L2G25B and 4-1BB were considered as possiblecandidates for the unidentified soluble mediators of Prystowsky et alwhich affected macrophage activities.

[0105] Isolation of human lymphokines and receptors homologous to L2G25Band 4-1BB. L2G25B and 4-1BB cDNA may be used as probes to isolate humanlymphokines or receptors homologous to these type clones. Each cDNA willbe radio-labeled and hybridized to human genomic DNA blot under variousstringency and washing conditions using standard laboratory techniquesknown to those skilled in the art.

[0106] The species difference in nucleotide sequences between human andmouse will determine the degree of homology by clone hybridizationexperiments. On the determination of the optimal hybridization andwashing conditions under which the probes detect a signal in the humangenomic DNA blot, then a human genomic library in lambda vector may bescreened with radio labeled L2G25B and 4-1BB. The hybridizing humanclones may then be isolated and the nucleotide sequences determined.

[0107] The genomic human clone corresponding to mouse clone L2G25 and4-1BB may then be used as a probe to survey human T cells which expressmRNA by RNA blot analysis. When the human T cells which express the RNAhomologous to L2G25B and 4-1BB are discovered and isolated, the RNA maythen be used to construct a cDNA library. Then the cDNA library may bescreened with the human genomic clone corresponding to L2G25B and 4-1BBand isolate the human cDNA clones corresponding to L2G25B and 4-1BB.

[0108] Plasmid p4-1BB may be used to grow the receptor 4-1BB. To do so:one must insert the cDNA of 4-1BB into an appropriate prokaryotic or aeukaryotic expression vector such as a Bovine Papilloma virus expressionvector; and transfecting that expression vector into mouse fibroblastsor other appropriate transfection hosts; and grow the then transfectedmouse fibroblasts in an appropriate culture media; and then purifyingthe lymphokine protein from the culture media.

[0109] cDNA in the form of plasmid p4-1BB in Ecoli NM 522 has beendeposited at the American Type Culture Collection under ATC No: 67825and will be available after this Patent Application issues.

[0110] An Inducible Receptor-Like Molecule. 4-1BB. is Expressed inInfiltrating Mononuclear Cells of Diabetic NOD Mice

[0111] The transcript of 4-1BB was inducible by concanavalin A in mousesplenocytes, T-cell clones, and hybridomas. The expression of 4-1BBtranscripts was inhibited by cyclosporin A. The 4-1BB mRNA was inducibleby antigen receptor stimulation but was not inducible by Il-2stimulation in the cloned T-cells (91). The 4-1BB cDNA encodes a peptideof 256 amino acids containing a putative leader sequence, a potentialmembrane anchor segment, and other features of known receptor proteins.Therefore, the expression pattern of 4-1BB resembles those of lymphokinemRNAs while the sequence appears consistent with those of receptorproteins.

[0112] The deduced amino acid sequence of 4-1BB is similar to tumornecrosis factor receptor (92). The 4-1BB protein is also a member of thenerve growth factor receptor super family, for the deduced amino acidsequence of 4-1BB predicts a cysteine-rich extracellular domain (93).Shaw et al. (94) mapped the amino acid sequence involved in thep56^(lck) binding in the cytoplasmic domains of T₄ and T₈ antigens. The4-1BB protein contains the consensus amino acid sequence which can bindto the p56^(lck) in the putative cytoplasmic domain.

[0113] The primary purpose of the research reported herein was tofurther develop our understanding of the biological function of thismolecule. In this context, an antiserum was prepared which recognizesthe 4-1BB protein and determined the protein expression in varioustissues of normal and pathologic mice.

[0114] As a diseased tissue, the pancreas of the diabetic NOD mouse waschosen for study. The NOD mouse has become an important model of Type I,or insulin-dependent diabetes mellitus (36, 37, 95). In both humans andNOD mice, an autoimmune pathogenesis is suggested by the presence oflymphocytic infiltrations in the pancreatic islets that appear to resultin selective β-cell destruction (96). The infiltrating T-lymphocytesappear to be activated by triggering agents, possibly auto-antigens,upon infiltration into the pancreas. As a step to further understandingthe biological functions of this molecule, the possibility that 4-1BB isexpressed in the infiltrating mononuclear cells in the pancreatic isletsof NOD mice was explored.

[0115] Materials and Methods

[0116] Cells. CTLL-R8, a mouse cytolytic T-cell line, (97) was grown inDMEM (Gibco Laboratories, Grand Islands, N.Y.) containing 100 units/mlof penicillin, 100 μg of streptomycin, 4 units/ml of rIL-2(Boehringer-Mannheim, Indianapolis, Ind.) and 10% FBS. RAW 264.7, amurine macrophage cell line (38-42), and EL-4, a mouse thymoma cell linewere cultured in DMEM, containing 10% FBS, 25 mM Hepes, 1 mM sodiumpyruvate, 100 units/ml of penicillin and 100 μg/ml of streptomycin.COS-1 cells were grown in DMEM containing 10% FBS, 100 units/ml ofpenicillin, and 100 μg/ml of streptomycin.

[0117] Antibody Preparation. Five oligopeptides representing differentregions of the deduced 4-1BB protein (4-1BBP) sequence were synthesized(Applied Biosystems, Foster City, Calif.). Two sequences, named 4-1BB-0and 4-1BB-11, stimulated the production of antibodies. The amino acidsequence of the oligopeptide 4-1BB-0 was a 12-mer from amino acids105-115 of the deduced 4-1BBP. Oligopeptide 4-1BB-11 was a 25-mer fromamino acids 133-157 of the deduced 4-1BBP. A tyrosine residue at theC-terminus of the oligopeptide 4-1BB-0 was added for labeling with[¹²⁵I] if needed. The peptides were conjugated to keyhole limpethemocyanin (KLH) using a heterobifunctional cross linker,m-maleimidobenzoyl-N-hydroxysuccinimide ester (98).

[0118] Rabbits were immunized with peptide-KLH (100 μg per dose)emulsified in Freund's complete adjuvant. The rabbits received oneintracutaneous injection in 4 foot pads and one intramuscular injectiontwo weeks apart. After two weeks, the rabbits received three consecutiveintravenous injections (50 μg per dose) without adjuvant. The serum wasobtained 5 days after the final injection, and the titer was measured byELISA using the peptide as the antigen.

[0119] Flow Cytometry. 1×10⁷ CTLL-R8 cells were incubated with preimmuneor IgG fraction of anti-4-1BB-rabbit serum on ice for 30 min. Cells werewashed three times in RPMI 1640 containing 5% FBS. Then the cells wereincubated on ice for 30 min with fluorescein isothiocyanate(FITC)-conjugated goat anti-rabbit immunoglobulin. The cells were washedagain with phosphate buffered saline (pH 7.4) containing 10% bovineserum albumin. Flow cytometry was performed using an EPICS 753 (Coulter)fluorescence-activated cell sorter.

[0120] Immunocytochemistry and Histology. The cells were cultured onsterile cover slides coated with poly-L lysine. For some experiments,CTLL-R8 cells were stimulated with concanavalin A (5 μg/ml) for variouslengths of time. The cells on coverslips were washed three times withphosphate-buffered saline (PBS), pH 7.4, for 5 min and fixed by treatingwith 3.7% formaldehyde in PBS at room temperature for 10 min and withmethanol at −20° C. for 4 min. Subsequently, the cells were treated withacetone at −20° C. for 1 min.

[0121] To prepare nonpathologic tissue slides, C57B1/6 mice wereperfused with 4% paraformaldehyde in PBS through the heart. Afterperfusion, organs were excised and cut in blocks of 1-2 mm thickness.The organs included the brain, heart, lung, thymus, liver, spleen,pancreas, and kidney. These tissue blocks continued to be fixed in 4%paraformaldehyde for 24 hrs. The tissue blocks were further cut with avibratome into slices of 40 μm thickness. Lung specimens were cut with acryostat after being frozen in Tissue-Tek O.C.T. (Miles Scientific,Naperville, Ill.).

[0122] Streptavidin-biotinylated alkaline phosphatase complex (ABC-Ap)(DAKO Corporation, Denmark) was used to stain cells or frozen sectionsof lung. A positive reaction was indicated by red staining with a fastred. Horseradish peroxidase-antiperoxidase (PAP) was used to stain thevibratome sections of other organs. The anti-4-1BB-0 rabbit serum wasused as primary antibodies and pre-immune rabbit sera were used ascontrols. Meyer's hematoxylin or methyl green was used for counterstaining.

[0123] To study the immuno-cytochemistry of the NOD mouse pancreas, thepancreas was fixed in Carnoy's B solution (75% ethanol and 25% glacialacetic acid) and was processed to make a paraffin block. The paraffinsections were deparaffinized, rehydrated in graduated alcohols andimmersed in PBS, while frozen sections were used after fixation in coldacetone. Appropriately diluted antibodies were incubated with thesections for 3 hrs at room temperature, followed by washing in PBS for 5min and incubation with FITC-labeled protein A (1:40 diluted in PBS with0.05% Evan's blue) for another 3 hrs at room temperature. Pictures weretaken with an Olympus BH-2 fluorescent microscope. Parallel withimmunofluorescence staining, the sections were stained with hematoxylinand eosin, allowing the grading of insulitis to be scored as describedelsewhere (99).

[0124] NOD Mouse. The colony of NOD mice was obtained from the SecondDepartment of Internal Medicine, Kobe University School of Medicine,Kobe, Japan. These animals were maintained on regular mouse chow and tapwater ad libitum at the University of Calgary. At the age of 10 weeks,the 5 NOD mice were treated with cyclophosphamide (Horner, Montreal,Quebec, Canada) at the dose of 150 mg/Kg body weight twice at 3-dayintervals. At the age of 26 weeks, they were sacrificed and pancreatawere excised.

[0125] Immunoblot Analysis. Cells grown in petri dishes were washed inPBS and lysed by adding TNE buffer (50 mM Tris HCl, pH 8.0, 1% NP-40, 2mM EDTA) on ice for 2 hrs. The TNE buffer contained the proteaseinhibitors aprotinin and leupeptin at 100 μg/ml each. The cell lysatewas harvested and centrifuged for 5 min. The supernatant containingapproximately 1 mg/ml of protein was denatured by boiling for 2 min in asample buffer consisting of 62.5 mM Tris HCl, pH 6.8, 10% glycerol, 1%SDS, 1% β-mercaptoethanol and 0.001% bromphenol blue. The proteins wereresolved on 12% SDS-PAGE (polyacrylamide gel electrophoresis) andtransferred electrophoretically onto an Immobilon membrane (Millipore,Bedford, Mass.). The membranes were blocked to prevent nonspecificantibody binding by incubating in 5% nonfat dry milk in TBST (50 mM TrisHCl, pH 7.4, 0.15 M NaCl and 0.05% Tween-20) for 1 hr at roomtemperature. The membranes were then treated with primary antibodies orprimary antibodies treated with oligopeptides at room temperature for 1hr. After four washes with TBST, the membranes were incubated with asecondary antibody against rabbit IgG(H+L)-alkaline phosphataseconjugate (Zymed, Inc., S. San Francisco, Calif.) at 1:100 dilution asrecommended by the manufacturer. The rabbit antiserum absorbed by anoligopeptide was prepared as follows. The rabbit antiserum was incubatedwith various concentrations of the oligopeptide (0, 0.1, 1.0, and 10μg/ml) in TBST buffer and 1% dry nonfat milk, then microcentrifuged at14,000 rpm for 20 min, and the supernatant was used as a primaryantibody after further dilution in TBST.

[0126] The reactive bands were visualized by incubating the membranewith chromogenic substrates, p-nitroblue-tetrazolinum chloride (NBT) and5-bromo-4-chromo-3-indolyl-phosphate (BCIP) (Bio Rad, Richmond, Calif.)in 0.1 M Tris, pH 9.5, 0.1 M NaCl, and 5 mM MgCl₂.

[0127] Construction of the Expression Plasmid of Truncated 4-1BB. Theputative extracellular domain of 4-1BB cDNA was amplified by polymerasechain reaction (PCR) (100). An Xho1 site was created at the 5′ end ofthe forward primer and a stop codon, (TAA), and an Eco R1 site werecreated in the reverse primer. The PCR product was digested with Xho1and Eco R1 and the 0.6 kb fragment was purified. The Xho1-Eco R1fragment (P4-1BBs) was inserted into the PXM vector (101).

[0128] Production of the Recombinant Truncated 4-1BB Protein. COS-1cells were grown to 30-50% confluency and were transfected with thetruncated 4-1BB in the PXM vector using the DEAE dextran method (102).Forty-four hours post transfection, the culture medium was replaced withserum-free medium (Opti MEM, Gibco Laboratories, Grand Island, N.Y.).The culture medium was harvested twice every 24 hrs. The proteins in theconditioned medium were precipitated with 4 volumes of acetone at −20°C. and resuspended in a mixture consisting of a chromatography buffer(50 mM Tris, pH 7.4, 0.15 M NaCl and 0.05% Tween −80), 5M urea and 1%β-mercaptoethanol. After the removal of undissolved particles by briefmicrocentrifugation, the supernatant was subjected to Sephadex G-200chromatography. The fractions that were reactive with rabbitanti-4-1BB-0 antiserum in Western blot analysis, were pooled. Thetruncated, thus soluble 4-1BB protein (4-1BBPs) was further enrichedthrough fractionation with Q-Separose column (Pharmacia Fine Chemicals)with a linear gradient of Nacl from 0.0 to 1.0M. The amino-terminalsequence of 4-1BBPs was determined by an automatic peptide sequencer PI2090 (Proton Instrument, Tarzana, Calif.) after the protein wastransferred to Immobilon-p (Millpore, Bedford, Mass.).

[0129] Northern Blot Analysis. Mouse organs were sliced into 1 mm thickpieces. A portion of each organ was stimulated by incubating in DMEMcontaining PMA (phorbol 12-myristate 13-acetate, 20 ng/ml) for 24 hrs. Aportion of the spleen was treated with concanavalin A (10 μg/ml) inDMEM. The remaining portion of each organ was incubated in DMEM plus 10%FBS without PMA or concanavalin A for 24 hrs. The tissues were frozen in−70° C. and pulverized in liquid nitrogen before extracting RNA. RNA wasextracted from the tissues and cells by the guanidinium-phenolextraction procedure (103). The RNA was fractionated on a 1.4%formaldehyde denaturing agarose gel, transferred to a Gene-Screen Plusmembrane, and hybridized to ³²P-labeled probes.

[0130] Results

[0131] Specificity of Antioligopeptide Antisera to 4-1BB and Expressionof 4-1BB Protein. The only information previously available on the 4-1BBwas the nucleotide sequence of the cDNA and the predicted amino acidsequence. In order to study the 4-1BB protein (4-1BBP), polyclonalantibodies were raised against oligopeptides representing five differentportions of the predicted 4-1BBP. To aid in proving that the putativeantisera contain antibodies which uniquely recognize the 4-1BBP, asearch for cell lines that express 4-1BB mRNA was made. CTLL-R8 cellsproduced a high level of 4-1BB mRNA while a macrophage cell line, Raw264.7, or unstimulated EL-4 cells did not produce detectable amounts of4-1BB mRNA (FIG. 111). FIG. 11 shows the expression of 4-1BB on RNA inCTLL-R8. Cytoplasmic RNA was prepared from mouse CTL line, CTLL-R8 (laneA), macrophage cell line, RAW 264.7 cells (lane B) and unstimulated EL-4cells (lane C). Ten micrograms of total RNA was fractionated on aformaldehyde/agarose gel, transferred to a Gene Screen Plus, andhybridized to 4-1BB cDNA probe. Arrows indicate the specific signals.Positions of 28S and 18S rRNA markers are indicated.

[0132] We then tested whether any of the antibodies recognized the threecell lines differentially. Two antisera among five tested had a positivereaction to CTLL-R8 cells while the antibodies did not stain RAW 264.7or EL-4 cells. One antioligopeptide antiserum, anti-4-1BB-0, stainedCTLL-R8 cells at a higher dilution (1:1600) than did the other one,anti-4-1BB-11. The staining pattern of CTLL-R8 cells revealed a diffusegranular distribution in the cytoplasm and uniform staining on the cellmembrane (data not shown). When the lysates of CTLL-R8, RAW 264.7 andEL-4 cells, were prepared and an immunoblot analysis performed with theanti-4-1BB-0 serum, a unique band of 40 kD was recognized in the CTLL-R8cells but not in the RAW 264.7 or EL-4 cells (FIG. 12).

[0133]FIG. 12 shows an immunoblot analysis of CTLL-R8 cell lysates withanti-4-1BB-0 serum. Lanes A and B contain approximately 20 μg of CTLL-R8cell lysate. Lanes C and D contain approximately 20 μg protein of RAW264.7 and EL-4 cell lysate, respectively. Lane A reacted with preimmunerabbit serum. Lanes B, C and D reacted with anti-4-1BB-0 serum (1:1600).The arrow indicates the 40 kD protein band on Lane B.

[0134] To prove further the specificity of the antioligopeptideantisera, an expression plasmid was prepared containing a truncated4-1BB cDNA. The membrane anchor and cytoplasmic domains were eliminatedfrom the 4-1BB cDNA. The truncated cDNA was inserted into the PXM vectorand expressed in COS-1 cells. The culture medium of the transfectedCOS-I cells was concentrated and fractionated by Sephadex G-200chromatography. An aliquot of each fraction was run on SDS-PAGE,transferred to the Immobilon membrane (Millipore, Bedford, Mass.), andtreated with the anti-oligopeptide antiserum, anti-4-1BB-0. A peak ofprotein was found which reacted with the anti-4-1BB-0. There was nodetectable amount of the 4-1BBP in the COS-1 cell lysate. The soluble4-1BBP (4-1BBPs) fraction was further purified through Q-sepharosecolumn. The molecular size of the 4-1BBPs was approximately 23 kD on a10% SDS-PAGE.

[0135] Next, a series of immunoblots was prepared containing the4-1BBPs. The blots were stained with unabsorbed or absorbed anti-4-1BB-0antiserum. FIG. 13 shows an immunoblot analysis of the 4-1BBPs. Lanes A,B, C, and D contain the cell culture supernatant of COS-1 cells whichwere transfected with truncated 4-1BB expression plasmids. Lane Areacted with unabsorbed anti-4-1BB-0 serum. Lanes B to D reacted withanti-4-1BB-0 serum absorbed by 0.1 μg/ml (lane B), 1 μg/ml (lane C) and10 μg/ml (lane D) of the 4-1BB-0 peptide. The arrow with the 23 kDindicates the bands seen on lanes A and B. FIG. 13, lane A, shows the4-1BBPs band (23 kD) from the COS-1 cell medium reacted withanti-4-1BB-0. The 4-1BBPs band gradually disappeared when theanti-4-1BB-0 was absorbed by the increasing amount of the oligopeptide4-1BB-0. As shown in FIG. 13, lane B, the antibodies to the 4-1BBprotein were not absorbed completely by 0.1 μg/ml of the 4-1BB-0peptide. However, when the concentration of 4-1BB-0 was increased to 1.0μg/ml (lane C) and 10 μg/ml (lane D), the anti-4-1BB-0 antibodies werecompletely absorbed, showing no 4-1BBPs bands.

[0136] The amino-terminal sequence of the purified 4-1BBPs wasdetermined. The sequence was Val-Gln-Asn-Ser-X-Asp. The amino acidsequence at positions 1, 2, 3, 4 and 6 was identical to that of themature 4-1BBP predicted from the cDNA sequence. Amino acid at position 5which is supposed to be Cys was not determined. These results indicatethat the deduced amino acid sequence and assignment of signal sequenceare correct. When the potential transmembrane domain was removed fromthe complete 4-1BB molecule, the protein was secreted. These resultssuggested that 4-1BBP was likely to be associated with the cellularmembrane as predicted by the primary structure.

[0137] The 4-1BBP expression was analyzed by flow cytometry and cellsorting using an EPICS 753 fluorescence-activated cell sorter (Coulter).FIG. 14 shows representative histograms of IgG fraction of anti-4-1BB-Orelated fluorescence intensity of CTLL-R8 cells. X-axis representsfluorescence intensity and Y-axis, cell numbers. A: Unsorted CTLL-R8cells stained with IgG fraction of anti-4-1BB-O. B and C: The stainedpopulation in A was separated from the rest of the cells, cultured for 8days, and stained with either preimmune (B) or IgG fraction ofanti-4-1BB-O (C). Flow cytometry and cell sorting were performed with anEPICS 753 fluorescence-activated cell sorter (Coulter).

[0138] When the CTLL-R8 cells were stained with IgG fraction of theanti-4-1BB-O rabbit serum, approximately 21% of the cells were labeled(FIG. 14A). Next, a sort for the 4-1BB⁺ cells was performed and thesecells were cultured in the presence of 20 units/ml of rIl-2. These cellswere cultured for 8 days before testing 4-1BBP expression by flowcytometry again. When these cells were stained with anti-4-1BB-O serum,98.3% of this cell population was labeled (FIG. 14C), while preimmuneserum stained the cells at background level (FIG. 14B). These resultsindicated that the 4-1BB protein was expressed on the cell surface andperhaps represented a receptor.

[0139] The Tissue Distribution of 4-1BB. RNA was extracted from tissuesof various organs and tested for the expression of 4-1BB mRNA. 4-1BBmRNA was detected in the spleen, kidney, and heart; but no RNA wasdetectable in the liver, adrenal gland, or pancreas. The RNA level wasmarkedly elevated when the spleen and heart were treated with PMA, butother organs did not show 4-1BB RNA induction after PMA treatment (FIG.15).

[0140]FIG. 15 shows the expression of 4-1BB RNA in mouse tissues. Thetotal RNA from the spleen (lanes A, B, and C), heart (lanes D and E),and kidney (lanes F and G), was fractionated on formaldehyde-denaturingagarose gel, transferred to Gene Screen Plus, and hybridized to a³²P-labeled 4-1BB cDNA probe. Each lane contains 20 μg of RNA. Lane A:unstimulated spleen RNA, lane B: concanavalin A-stimulated spleen RNA,lane C: PMA-stimulated spleen RNA, lane D: unstimulated heart RNA, laneE: PMA-stimulated heart RNA, lane F: unstimulated kidney RNA, and laneG: PMA-stimulated kidney RNA. Positions of 28S and 18S rRNA markers areindicated. An arrow indicates the specific hybridization signal.

[0141] The 4-1BBP was detected in the medullary tubules and medullaryrays of the kidney (data not shown). A small number of mononuclear cellsin the alveolar septae of the lungs and some lymphocytes in the spleenshowed weak staining. The pancreas, liver, testes, and ovary expressedneither 4-1BB mRNA nor 4-1BBP. The detailed description of tissuedistribution of the 4-1BBP will be published elsewhere.

[0142] 4-1BB Expression in Infiltrating Mononuclear Cells. In serialsections of the pancreata, 40 islets were observed afterhematoxylin-eosin staining. FIG. 16 shows the histology of NOD mousepancreata and immunofluorescent staining of islets showing differentstages of insulitis. Pancreas sections of A, C, and E were stained withthe standard hematoxylin and eosin staining technique, while those of B,D, and F were stained with anti-4-1BB-0 serum and FITC-conjugatedprotein A. B, D, and F are the corresponding islets to A, C, and E,respectively. A and B: early-stage insulitis, C and D:intermediate-stage insulitis, and E and F: late-stage insulitis. Notethe strong immunofluorescent staining in the mononuclear cells at theperiphery of islets showing the early stage of insulitis, while thestaining is not apparent in the islets showing the late stage ofinsulitis.

[0143] Among those islets, 10 showed no insulitis and 12 showed signs ofearly insulitis in which lymphocytes had infiltrated only to theperiphery of each islet (FIG. 16A). The remaining 18 islets showedintermediate or late stage insulitis in which the lymphocytes hadinfiltrated into the islets and showed signs of islet destruction (FIGS.16C and 6E). The serial sections were then stained with anti-4-1BB-0.None of the 10 intact islets showed 4-1BB expression (Table 3). However,mononuclear cells accumulated at the periphery of the islets werestained with the anti-4-1BB-0 serum in the early stage of insulitis(FIG. 16B). All 12 of the early insulitis islets demonstrated the samestaining pattern (Table 3). 4-1BB expression diminished gradually in themononuclear cells during the intermediate to late stages of insulitis(FIGS. 16D and 6F). Seven of the 18 islets of intermediate to late stageinsulitis showed weak staining in the infiltrating mononuclear cells,and the remaining 11 islets showed no staining. These results indicatethat the 4-1BB protein is most likely associated with T-cell functionsduring the early phase of activation. Such an early expression of 4-1BBprotein is well corroborated with the finding that 4-1BB mRNA wasdetectable as early as 30 min after the T-cells are stimulated withlectins or antibodies to T-cell receptors. TABLE 3 Expression of the4-1BB protein in the insulitis lesion of NOD mice according to the gradeof insulitis. 4-1BB Expression Stage of Insulitis No. Strong Weak NoneIntact islets 10  0 0  6 Early 12 12 0  0 Intermediate/Late 18  0 7 11

[0144] The insulitis lesion was arbitrarily classified as early,intermediate, and late-stage insulitis according to the morphologicalcriteria as follows. Early insulitis was defined as an accumulation ofmononuclear cells at the periphery of or just within the islet. Inintermediate insulitis, the mononuclear cells infiltrated the center ofthe islets but left the architecture of the islets relativelywell-preserved. In late insulitis, the architecture of islets wasdistorted, and infiltration by mononuclear cells was markedly increased.The expression of the 4-1BB protein was scored as strong, weak or none.The strong expression indicates that more than 50% of the infiltratingmononuclear cells were positive (FIG. 15B), while weak expression meansonly a few cells were positive (FIG. 15D).

[0145] Discussion

[0146] The 4-1BB cDNA was isolated based upon its preferentialexpression in T-cells. These experiments demonstrated that 4-1BB isexpressed constitutively in renal medullar cells and that expression isinduced in the spleen and heart by PMA treatment. In the spleen, T-cellsare likely to respond to PMA or concanavalin A and are probablyresponsible for the increase of 4-1BB mRNA. However, it is not knownwhich cells in the heart respond to PMA to produce 4-1BB mRNA.

[0147] The earlier studies showed that the protein backbone of thenatural 4-1BBP is composed of 233 amino acids with an Mr of 25 kD. Thisprotein therefore must undergo an extensive post-translationalmodification to be 40 kD protein. The deduced 4-1BBP contains both N-and O-glycosylation sites. Occasionally anti-4-1BB-0 serum recognizes 38and 34 kD extra bands, which were believed to represent 4-1BBP withdifferent degrees of glycosylation. The protein backbone of truncated4-1BB is composed of 162 amino acids with an Mr of 18 kD. Since thetruncated form produced by COS-1 cells resolves at 23 kD, this form of4-1BB also undergoes post-translational modification. In fact, when the4-1BBPs were produced in baculoviral expression system 4-1BBPsconstituted three bands of 18 ID, 20 kD and 23 kD which, are believed torepresent 4-1BBPs with different degrees of glycosylation (unpublishedobservation).

[0148] The primary structure of 4-1BB, the flow cytometric analysis of4-1BBP expression, and the secretion of 4-1BBPs indicate that the 4-1BBPis associated with the cellular membrane. Why a certain population ofCTLL-R8 cells expressed 4-1BB constitutively was not known. Perhaps agroup of cells changed its properties during the long in vitromaintenance. CTLL-R8 was a cloned CTL which lost killing activity in thecourse of in vitro maintenance.

[0149] To determine more about the functions of this receptor-likemolecule, it was decided to find the ligands. The truncated, thussecretory and soluble, 4-1BBPs have been value in determining thefunction of 4-1BB. It was postulated that if the 4-1BBPs competed forthe membrane form of 4-1BB with the specific ligand, the 4-1BBPs mightfunction as a specific inhibitor.

[0150] It was believed that the infiltrating mononuclear cells which arestained by anti-4-1BB-0 antibodies are activated T-lymphocytes becauseonly splenocytes stimulated with Con A or anti-TCR antibody expressed4-1BB mRNA. Detailed studies on the surface markers of infiltratingcells that are stained by anti-4-1BB-0 antibodies were underway. It isinteresting that 4-1BBP is expressed in the early phase of insulitis anddisappears when T-cells infiltrate into islets. It was postulated that4-1BBP might be a receptor which transduces signals from the membrane tothe nucleus necessary for the immediate early phase of inflammation orantigen recognition. This is in contrast to the expression of othermolecules such as perforin (104). Perforin, a potential molecule fortissue damage (91), is produced when T-cells infiltrate the islets. Theexpression of perforin in the early stage of insulitis was almostundetectable (unpublished observations).

[0151] The dynamic functions of T cells in a successful immune systemare accomplished through mediators which are produced when T cells areactivated. These mediators are in the form of cell surface receptors andsoluble secretory molecules. Identification of new mediators and thedemonstration of their functions can lead to the discovery of unknownfunctions of T cells and to the development of ways to manipulate theimmune system in the treatment of disease.

[0152] Expression of a Novel T-Cell Molecule, 4-1BB, in the Brain

[0153] The responses of both the immune and nervous systems toenvironmental change are mediated by soluble secretory proteins andreceptors. Although, to date few biological molecules which are sharedby these systems have been identified, the linking of the immune andnervous systems has been the focus of much speculation and hadstimulated widespread interest.

[0154] A series of T-cell subset-specific cDNAs were cloned from clonedhelper and cytolytic T-lymphocytes by employing a modified differentialscreening procedure. The transcript of one of the clones, 4-1BB, wasdetected in the T-lymphocytes when the T-cells were activated by eitheran antigen receptor stimulation or concanavalin A (Con A). This inducedexpression was inhibited by cyclosporin A. The predicted 4-1BBPcontained an unusually large number of cysteins. These residues werearranged with a spacing similar to those in several groups of proteinsincluding the epidermal growth factor receptor. The potential 4-1BBsequence showed similarities with the sequences of the tumor necrosisfactor receptor and the nerve growth factor receptor (97). The receptorfeature of 4-1BB and the resemblance to the nerve growth factor receptorprompted us this investigation with the brain. Using Northern blotanalysis of mRNA and immunocytochemistry for detecting 4-1BB protein(4-1BBP), and it was unexpectedly found that the 4-1BB protein has ahigh and constitutive expression in the brain, an organ which containsabundant receptor elements and is ontogenically separate from the immunesystem. The following report deals with the expression of 4-1BB in thenervous system and focuses on its distribution in the brain and in theperipheral nerves.

[0155] Method & Materials

[0156] Northern Blot Analysis. Mouse organs were sliced into pieces of 1mm thickness, and a portion of each organ was incubated in PMA (PhorbolMyristic Acetate, 20 ng/ml) containing Dulbecco's minimum essentialmedium (DMEN, GIBCO) and 10% fetal bovine serum (FBS) for 24 hrs. Aportion of spleen was treated with ConA (10 μg/ml) in DMEM and 10% FBS.The remaining portion of each organ was incubated in DMEM and 10% FBSwithout PMA or ConA for 24 hrs. The tissues were frozen at −70° C. andpulverized in liquid nitrogen before extracting RNA. RNA was extractedfrom the tissues and cells by guanidium-phenol extraction procedure (86,87). The RNA was fractionated on a 1.4% formaldehyde denaturing agarosegel, transferred to a Gene Screen Plus membrane, and hybridized to a³²P-labeled 4-1BB cDNA probe.

[0157] Antibody Preparation. An oligopeptide representing amino acids105-115 of the deduced 4-1BBP sequence was synthesized (AppliedBiosystem). The sequence was NH₂-CRPGQELTKSGY-COOH. A tyrosine residueat the C-terminus of the peptide was added for possible radioactivelabeling with [¹²⁵I]. The peptide was conjugated to keyhole limpethemocyanin (KLH) with a heteroblifunctional cross linker,m-maleimidobenzoyl-n-hydroxysuccinimide ester (88, 107).

[0158] Rabbits were immunized with peptide-KLH (100 μg/dose) emulsifiedin Freund's Complete adjuvant. In two week intervals they received oneintracutaneous injection in each of four foot pads and one intramuscularinjection. After two weeks, the rabbits received three consecutive I.V.injections without adjuvant. The serum was obtained five days after thefinal injection and the titer was measured by ELISA using peptide as theantigen. The specificity of these antibodies to 4-1BBP (anti 4-1BB-O)was shown in pervious studies.

[0159] Immunocytochemistry. the procedure for immunocytochemistry waspublished previously (108). In brief, C57B1/6 mice were perfused withformaldehyde made fresh intracardially under deep anesthesia from 4%paraformaldehyde and 0.1 M phosphate-buffered saline (PBS). Brains andmuscle from the gluteal region were then removed, left in the samefixative overnight, and sliced into 40 um sections forimmunocytochemical staining. The 4-1BB-(1:200) antiserum was used forpositive staining, and antiserum preabsorbed with 4-1BB (10 μg/ml) wasused as control. The Stemberger's peroxidase-anti-peroxidase (PAP)indirect-enzyme method was used for staining. The PAP reaction was donewith 0.003% H₂O₂ and 0.05% 3′3-diaminobenzidine. The primary andsecondary antibodies were diluted with PBS containing 0.2% Triton-X100and 1% normal sheep serum. The primary antibodies were incubatedovernight and the secondary antibodies were incubated for one hour.Rabbit antiserum against purified mouse laminin (E.Y. Labs, San Mateo,Calif.) was used as a control antiserum.

[0160] Results

[0161] Similarity of 4-1BBP to other Known Proteins. The 4-1BBP shows asimilarity to the nerve growth factor receptor, the tumor necrosisfactor receptor, CD40, and the Shope fibroma virus T2 proteins asdescribed by Smith et al (97). A search was made for the proteins whichcontain regions similar to those of 4-1BBP and found two other potentialproteins which were encoded by seven in absentia (sina) and by DG17.Sina genes are required by the R7 photoreceptor cell of the Drosophilaeye for correct R7 cell development (109). The N-terminal cysteine-richregion of the sina protein is extensively similar to the 4-1BBP (FIG.17) and is also similar to the protein product of the Dictyostelium DG17gene, whose expression is specifically induced during aggregation bycAMP (110).

[0162]FIG. 17 shows a comparison of the 4-1BBP amino acid sequence withthe amino acid sequence in sina of Dorsophila and DG17 of Dictyostelium.The amino acids which are shared are boxed. Numbers represent thepositions of the left-most residues relative to the N-terminus. Gaps (−)are introduced to allow for maximal alignment.

[0163] This region forms the pattern of C—X₂—C—X₉—H—X₃—C—X—C; and thecysteines and histidine are conserved in a similar space in 4-1BB, sina,and DG17 proteins. Ten of 24 amino acids between the 4-1BB and sinaproteins are identical. Between 4-1BB and DG17 proteins, 11 of 24 aminoacids are identical, and 3 of 24 are conservative substitutions. Theconserved pattern suggests that these amino acids are functionallyimportant.

[0164] 4-1BB mRNA Expression. As shown in FIG. 18, 4-1BB RNA wasdetected in the brain (lanes A and B), and heart (lanes C and D)_and thespleen (lanes G, H. and I) while 4-1BB RNA was not detected in thepancreas (lanes E and F). FIG. 18 shows a northern blot analysis ofkidney and brain RNA. Total cytoplasmic RNA from brain (A and B), heart(C and D) and spleen (E, F, and G) were fractionated on 1.2%formaldehyde denaturing agarose gel and hybridized to [³²P]-labeled4-1BB cDNA probe. Lane A: unstimulated brain RNA; Lane B: PMA-stimulatedbrain RNA; Lane c: unstiumlated heart RNA; Lane D: PMA-stimulated heartRNA; Lane E: unstiumlated spleen RNA; Lane F: PMA-stimulated spleen RNA,and Lane G: ConA-stimulated spleen RNA. Positions of 28S and 18S areindicated by 28 and 18, respectively. The arrow I indicated 4-1BB RNAfrom brain and the arrow II indicates 4-1BB RNA from spleen and heart.

[0165] 4-1BB RNA was inducible in the heart and the spleen (lands D andH) by PMA, and by ConA in the spleen (lane I), but was not inducible inthe brain. The size of the Brain 4-1BB RNA (FIG. 18, Arrow II) issmaller than that of 4-1BB NA's from other tissues (FIG. 18, Arrow I).In addition, the mRNA level in the brain is lower than that in othertissues. Such a result is surprising since this protein is detected at ahigh level in the brain. This may indicate that the 4-1BB m RNA has along half-life and may undergo several rounds of translation.

[0166] Immunocytochemistry: a) General distribution of 4-1BBimmunoreaction in the brain. Brain tissue (FIGS. 19 and 20) exhibitedthe most intense 4-1BB immunoreactive staining of all tissue examined,including liver, kidney, and muscle. Generally, dark staining productsare densely distributed in the gray matter where neuronal soma,dendrite, and fiber terminals reside (FIG. 18). Except the neuronal somaand distinct fiber bundles, most of the gray matter was stained. In thebrain region, where only glial cells accumulate, no staining wasidentified. Thus, definitive localization of 4-1BB-like protein in thebrain was strictly limited to the gray matter. No staining was observedin the major fiber bundle such as corpus callosum, cingulum bundle,internal capsule bundle, fimbria-formix, medial longitudinalfascicularis, or medial forebrain bundle.

[0167]FIG. 19 shows 4-1BB immunostaining in the cortex (a, b, and d)striatum (a, d, and e) at progressively enlarged magnifications. 4-1BBstaining is seen in the majority of gray matter but is absent in neuronand glial cell bodies, white matter, and in fiber bundles (see corpuscallosum, CC in a, and internal capsules, IC and d and e). Granularshape of 4-1BB staining (arrows) is seen around the neuronal bodies(stars in c and e) but not around glial bodies within fiber bundles (dand e). The 4-1BB positive granules in the striatum (e) resemble thedopamine terminals.

[0168]FIG. 20 shows distinct 4-1BB immunopositive reaction in thecerebellum at three progressively enlarged magnifications (a, b, and c).The 4-1BB positive reaction is most intense in molecular layer (M),lighter on Purkinje layer (P), and forms glomerulus-like patches(arrows) in the granular layer (G). It is generally absent in the cellbodies of Purkinje, granular layer (G). It is generally absent in thecell bodies of Purkinje, granular neurons, and glial cells. It iscompletely absent in axons-in the fiber bundle in the white matter (W).The majority of the 4-1BB stainings are accumulated in the terminalregions, where synapsis occurs. It was negatively stained when theantiserum was preabsorbed with antigen, 4-1BB peptide (d). Scales: a=200um, b,d,=100 um, and c=30 um.

[0169] High magnification (100× oil lens) of light microscopicphotographs showed that these distinctly stained 4-1BB immunoreactiveproducts are granular in shape with a size of 0.46-0.55 um. These 4-1BBgranules seem to reside among neurons and on the surface of neuronalsoma (FIG. 19) and perhaps also on the dendrites/proximal axons. Thedensity of the 4-1BB-like granules varies from region to region, anddensity distribution is often coincident with that of neuronal fiberterminals. The most intense 4-1BB positive staining was see in thestriatum and closely resembles dopamine-fiber terminals in the straitum(FIG. 19d,e).

[0170] Immunocytochemistry: b) Specific Regions. Unique distribution of4-1BB-like staining was observed in a number of brain regions. In thecortex, 4-1BB-like granules were packed in the molecular layer and weredistributed with sparse accumulation in layers II to VI (FIG. 19a,b,c).They were relatively homogeneous among layers except in the frontalcortex, where a band of dense immunoreaction was located in layer IV,and in the temporal cortex, where a dense but relatively narrower bandwas located within the molecular layer (not shown).

[0171] The most densely distributed immunoreactive granules were in thestriatum (FIG. 19d) and in the molecular layers of the cortex (FIG.19a,b,c), hippocampus, and cerebellum (FIG. 20). In the cerebellum, thestaining pattern was unique in that the 4-1BB-like granules were denselypacked in the molecular layer, loosely distributed in the Purkinjelayer, accumulated as islands in the sea of granules and Golgi cells inthe granular layer, and were almost blank in the fiber bundle area (FIG.20a,b,c, and d). The 4-1BB-like granules formed islands and did not seemto border cell bodies as seen in other brain regions. The morphologystrongly resembled the glomerulus in the cerebellum. The similarisland-like accumulation of 4-1BB-like immunoreactive granules alsoexisted in the nucleus of the stria medularis (not shown).

[0172] Discussion

[0173] 4-1BB, although expressed constitutively in the brain, isproduced in the t_cells only when t-cells are activated; therefore, themain site of in-vivo function may be the brain and not T-lymphocytes.Such a common expression of 4-1BBP may provide a clue to thecommunication pathway between the immune and nervous systems. 4-1BBPcontains a putative zinc finger structure of the yeast elF-2B protein(57) and shared a conserved region with the sina and DG17 proteins. Thesina protein is localized in the nucleus, suggesting that it has aregulatory function in cells.

[0174] The 4-1BBP has been detected at the cellular membrane, cytoplasm,and the nuclear membrane (unpublished observation). The fact that theamino acid sequence of 4-1BB contains features like a zinc finger motif,a nuclear protein, and a receptor domain may indicate that 4-1BB cancommute from the cell surface to the nucleus. Determining the ligand of4-1BBP and its functions is, therefore, critical to further defining thefunctions of 4-1BBP.

[0175] The reasons for that the size of brain 4-1BB RNA is smaller thanthat of 4-1BB RNA from other tissues are not known. It is possible thatbrain expresses in RNA species similar to 4-1BB sequence, not the 4-1BBgene transcripts. The brain molecules detected by anti-4-1BB-Oantibodies, therefore, may well be a cross-reacting protein whichcontains certain antigen epitopes similar to those of 4-1BBP.Nevertheless, the identification of the brain molecule detected byanti-4-1BB-O antibodies would be important because of the uniquepatterns of expression.

[0176] The mRNA expression and abundant immunostaining of 4-1BB-likeprotein in the brain indicate that 4-1BB is actively expressed andconstitutes a significant component of the brain. Such abundantexpression is not seen in the muscle or liner. Much of the evidenceindicated that 4-1BB can be a receptor or nerve terminal in the brainand peripheral nervous system: a) morphological examination of theimmunostain shows that the 4-1BB-like protein is located in the graymatter, particularly in the regions of dendrites, fiber terminals, andaround the cell body of the brain, while being almost entirely absentfrom the white matter where synapses do not occur; b) the granule-shapemorphology resembles the fiber terminals of GABAergic neurons on thesubstantial nigra, dopaminergic neurons on striatum, and synapsin, asynaptic membrane specific protein in the brain; c) while generallyabsent in the neuronal body and completely absent in and around glialcells, the protein was densely accumulated in many terminal regions ofthe brain; d) variable densities in regions such as cerebellum,striatum, and cortex coincided with dense fiber terminals; e) peculiarrosette patterns in the cerebellum and stria terminalis morphologicallyresembled the glomerulus, a specific synaptical complex in thecerebellum.

[0177] Recently, two neurotrophic factors, brain-derived neurotrophicfactor (BDNF) (57), and neurotrophin-3 (NT-3) (112), were identified inaddition to the nerve growth factor (NGF). These three factors closelyresembled one another with 57 of the 119 residues (48%) are shared byall three proteins. Six cysteines found in these factors were absolutelyconserved, and the regions of greatest similarity were mainly clusteredaround these cysteine residues. If BDNF and NT-3 utilize their ownreceptors, the receptor might have similar structural properties to NGFreceptor. The structural similarity of 4-1BB to the NGF receptorallowing a suspicion that 4-1BBP actually encodes one of such known oryet unknown neurotrophic factors.

[0178] Inducible T Cell Antigen 4-1BB: Analysis of Expression andFunction

[0179] 4-1BB is an inducible receptor-like protein expressed in bothcytolytic and helper Th cells. The 4-1BB mRNA was expressed inPMA-treated spleen and heart with constitutive expression detected inthe kidney. The optional induction of 4-1BB mRNA required both PMA andionomycin stimulation indicating that protein kinase C activation andincreases in intracellular Ca² were required for its expression. 4-1BBwas categorized as an early activation gene since the protein synthesisinhibitor, cycloheximide, blocked the induction of 4-1BB mRNA. Amonoclonal antibody, 53A2, was prepared against recombinant soluble4-1BB and used to characterize this molecule. 4-1BB was a 30 kDaglycoprotein and appeared to exist as both a monomer and a 55 kDa dimeron the cell surface. The 4-1BB protein may be post-translationallymodified since its predicted backbone is 25 kDa. FACS analysis indicatedthat 4-1BB was inducible and expressed on the cell surface of activatedsplenic T cells and thymocytes. Crosslinking of 4-1BB onanti-CD3-stimulated T cells with 53A2 resulted in a dramatic enhancementof T cell proliferation. This suggests that 4-1BB may function as anaccessory signaling molecule during T cell activation.

[0180] Recently, a number of cysteine-rich receptor proteins have beendescribed and named as the nerve growth factor receptor (NGFR)super-family. (92) At present, the members of the NGFR super-familyinclude NGFR (45); B cell antigen CD4O (46); the MRC OX-40 antigen (47),which is a marker of activated T cells of the CD4 phenotype; tworeceptors for tumor necrosis factor (TNF) called TNFR-I and TNFR-Il,which are found on a variety of cell types (48,91); SFV-T2 (121); anopen reading frame (ORF) in the Shope fibroma virus which later wasidentified as a virally-encoded, soluble form of TNFR-I; a T cellsurface antigen, CD27, which may be involved in T cell activation (49);Fas, a cell surface antigen that can mediate apoptosis (50); and SalF19R, an ORF in the Shope sarcoma virus (51), as well as 4-1BB, which isthe most distantly related member. The newest member is CD3O, aHodgkin's lymphoma antigen which may play a role in tile regulation ofcellular growth and transformation (52). Members of the family arecharacterized by the presence of three to six patterns of cysteine-richmotifs which consist of about 40 amino acids in the extracellular partof the molecule. These molecules contain a hinge-like region immediatelyadjacent to the transmembrane domain. This region is characterized by alack of Cysteine residues and a high proportion of Ser, Thr and Pro,which are likely to be glycosylated with O-linked sugars.

[0181] 4-1BB contains other interesting features in its cytoplasmicdomain. Those include 1) two runs of acidic amino acids; 2) a potentialp56^(lck) binding site; 3) five consecutive glycines at the carboxylterminus; and 4) four potential phosphorylation sites—1 tyrosine, 2threonine, and 1 serine. It is especially interesting that 4-1BBcontains a potential p56^(lck) binding site, —C—R—C—P—. The consensussequence of p56^(lck) binding site is —C—X—C—P— in the CD4 and CD8molecules (93).

[0182] To further understand the biologic function of 4-1BB, a mAb,53A2, was prepared which recognizes the 4-1BB molecule. This reagent wasused to characterize biochemical and expression properties of 4-1BB aswell as assess the role of 4-1BB signaling in T cells.

[0183] Materials and Methods

[0184] Cells

[0185] CTLL-R8 a mouse cytolytic T-cell] line, was grown in DMEM (GibcoLaboratories, Grand Islands, N.Y.) containing 100 units/ml ofpenicillin, 100 pg/ml of streptomycin, four units/ml of rIL-2(Boehinger-Mannheim, Indianapolis, Ind.) and 10% fetal bovine serum(FBS). F1 is a CD4⁺, I-A^(d) reactive T cell-clone which was generouslyprovided by Dr. Scott Bryson, University of Kentucky. This clone wasisolated from DBA/2 mice that had syngeneic graft-versus host disease.The clone was maintained in PRMI 1640 (Gibco Laboratories) containing10%FBS, 50micromoles 2-ME, and antibiotics (coplete medium). Rat Con Asupernatent was added at 30%. Spadoptera frugiperda (Sf-21), an insectcell line was grown in synthetic serum free medium, Ex-cell 400 (JRHBiosciences) containing antibiotics at 27° C. (53).

[0186] Preparation of Splenic T Cells

[0187] The resting murine splenic T cells were enriched by nylon wooland Percoll gradient centrifugation (54, 55). Briefly, the spleen cellsof female Balb/c mice (Harlan, Indianapolis, Ind.) were adjusted to1.5×10⁸/ml in RPMI 1640 containing 2% fetal bovine serum (FBS). A 10 mlnylon wool column was prewashed with phosphate buffered saline, pH 7.4(PBS) followed by RPMI 1640 containing 2% FBS, and incubated at 37° C.for 20 min before use. The spleen cells were loaded onto the column andincubated at 37° C. for 30 min. The column was washed with the abovemedium. The eluted cells subsequently were fractionated by centrifugingthe cells at 2000×g at 4° C. for 30 min or 50-100% Percoll stepgradient. The resting T cell fraction was recovered from the interfacebetween the 50% and 80% Percoll. The enrichment of T cells were examinedwith EPICS Profile Analyzer (Coulter Corporation); −91% were Thy 1.2⁺,−52% were L3T4⁺ and −24% were Lyt 2⁺. These cells were resting sincethey exhibited a uniform low degree of forward angle light scatter.

[0188] Thymocyte Preparation

[0189] Thymuses from 6-8 week old Balb/c mice were aseptically removedand teased into a cell suspension. Thymocytes were washed twice in RPMI1640 containing 2% fetal bovine serum (FBS).

[0190] Production of Recombinant 4-1BB Protein in Sf-21 Cells

[0191] To construct a plasmid that expresses extracellular portion of4-1BB, the 30 putative extracellular domain of 4-1BB cDNA (89) wasamplified by polymerase chain reaction (PCR) (99). An XhoI site wascreated at the 5′ end of the forward primer and a stop codon, (TAA), andan EcoR1 site were created in the reverse primer. The PCR product wasdigested with XhoI and EcoR1 and the −0.6 kb fragment was purified. TheXhoI-EcoR1 fragment (4-1BBS) was inserted into the PEV-55 vectors (53),generating PEV-55-4-1BBS. The sequence of the forward primer was5′-ACCTCGAGGTCCTGTGCATGT-GACA-3′ and that of the reverse primer was5′-ATGAATTCTTACTGCAGG-AGTGCCC-3′.

[0192] To express the entire 4-1BB protein, 1.2-kb cDNA fragment(4-1BBL) that contains all the coding sequence of 4-1BB was inserted inthe EcoR1 site of PVL 1392 vector (a kind gift from Dr. Max Summers),generating PVL 1392-4-1BBL.

[0193] 4-1BBS as well as 4-1BBL were transferred from these plasmids toAutographa Californica nuclear polyhedrosis virus (AcNPV) genome bycotransfection into Sf-21 cells as described (53), Tenocclusion-negative viruses for each construct were plaque-purified.AcNPV-4-1BBS and AcNPV-4-1BBL recombinant viral stocks were grown inSf-21 cells in serum-free Ex-cell 400 medium.

[0194] Rabbit Polyclonal Antiserum Against 4-1BB Oligopeptides

[0195] Five oligopeptides representing different regions of the deduced4-1BB protein (4-1BBP) sequence were synthesized (Applied Biosystems,Foster City, Calif.). Two sequences, named 4-1BB-0 and 4-1BB-11,stimulated the production of antibodies. The amino acid sequence of theoligopeptide 4-1BB-0 was a 12-mer from amino acids 105-115 of thededuced 4-1BBP (89). Oligopeptide 4-1BB-11 was a 25-mer from amino acids133-157 of the deduced 4-1BBP (2). A tyrosine residue at the C-terminusof the oligopeptide 4-1BB-0 was added for labeling with [¹²⁵I] ifneeded. The peptides were conjugated to keyhole limpet hemocyanin (KLH)using a heterobifunctional cross linker,m-maleimidobenzoyl-N-hydroxysuccinimide ester (97). Rabbits wereimmunized with peptide-KLH (100 pg per dose) emulsified in Freund'scomplete adjuvant. The rabbits received one intracutaneous injection ineach of 4-foot pads and one intramuscular injection two weeks apart.After two weeks, the rabbits received three consecutive intracutaneousinjections (50 μg per dose) without adjuvant. The serum was obtained 5days after the final injection, and the titer was measured by ELISA.

[0196] Purification of the rs-4-1BBP

[0197] The serum-free culture supernatants of Sf-21 cells infected byAcNPV-4-1BBS were concentrated by ammonium sulfate precipitation (60%saturation). The precipitate was dissolved in a buffer containing 40 mMTris-HCl (pH 7.8), 50 mM NaC1 and 0.02% NaN₃ and dialyzed in the samebuffer. The sample was subsequently fractionated by Sephacryl S-300 inthe above buffer. The fractions containing 4-1BBS were identified byWestern blotting with anti-4-1BB-0 antibodies. rs-4-1BBP containingfractions were pooled and concentrated by vacuum dialysis. The samplewas again fractionated by Q-Sepharose, an anion exchange columnchromatography. rs-4-1BBP-containing fractions were pooled, dialyzedagainst 5 mM sodium phosphate buffer pH 7.2 and were furtherfractionated by hydroxylapatite column. Finally, thers-4-1BBP-containing samples were equilibrated with 50 mM sodiumphosphate buffer (pH 7.2) and loaded on Sepharose. A linear gradientfrom 0 to 0.5 M NaC1 was used to elute absorbed proteins. Thers-4-1BBP-containing fractions were pooled and concentrated by vacuumdialysis.

[0198] Production of Anti-41BB mAb

[0199] Eight week-old Sprague-Dawley rats were immunized with 50 μg ofrs-4-1BBP emulsified in Titermax (cytRX). (subcutaneously) twice at atwo-week interval. A third (intracellular) injection was given two weeksafter the last immunization. Three days after the final injection therat spleen was removed. Spleen cells were fused with SP2/0 mouse myelomacells and cultured according to the standard method (56). ELISA was usedto screen for the rs-4-1BBP-reacting clones. Seventeen clones wereisolated and subcloned. One clone 53A2 (IgG¹) was characterized and isdisclosed herein. The monoclonal antibody was purified from culturesupernatant by affinity chromatography on protein G-Sepharose(Schleicher-Schnell).

[0200] Immunoblot Analysis

[0201] Cells were washed in PBS and lysed by adding TNE buffer (50 mMTris HCl, pH 8.0, 1% NP-4-0, 2 mM EDTA) on ice for 2 hrs. The TNE buffercontained the protease inhibitors, aprotinin and leupeptin at 100 μg/mleach. The cell lysate was harvested and centrifuged at 10,000×g for 10min at 4° C. The supernatant containing approximately 1 mg/ml of proteinwas denatured by boiling for 2 min in a sample buffer consisting of 62.5mM Tris HCI, pH 6.8, 10% glycerol, 1% SDS, 1% β-mercaptoethanol and0.001% bromphenol blue. The proteins were resolved on 12% polyacrylamidegel with SDS and transferred electrophoretically onto an Immobilonmembrane (Millipore, Bedford, Mass.). The membranes were blocked toprevent nonspecific antibody binding by incubating in 5% nonfat dry milkin TBST (50 mM Tris HCl, pH 7.4, 0.15 M NaCl and 0.05% Tween-20) for 1hr at room temperature. The membranes were then treated with primaryantibodies at room temperature for 1 hr. After four washes with TBST,the membranes were incubated with a secondary antibody against rabbit orrat IgG(H+L)-alkaline phosphatase conjugate (Zymed, Inc., S. SanFrancisco, Calif.) at 1:100 dilution. The reactive bands were visualizedby incubating the membrane with chromogenic substrates,p-nitrobule-tetrazolinum chloride (NBT) and5-bromo-4-chromo-3-indolyl-phosphate (BCIP) (Bio Rad, Richmond, Calif.)in 0.1 M Tris, pH 9.5, 0.1 M NaCl, and 5 mM MgCl₂.

[0202] Immunoprecipitation of Cell Surface 4-1BB

[0203] F1 cells were labeled with [³⁵S] cysteine (Amersham), at aconcentration of 0.1 mCi/ml for 14 hr in a cysteine-free RPMI 1640 and5% dialyzed-FBS. Normal and anti-CD3-stimulated F1 cells were washedtwice with PBS and resuspended in PBS containing 0.2% BSA and 0.1% NaN₃.To detect the cell surface 4-1BB antigen, cells were first incubatedwith 10 μg of 53A2 or unrelated rat IgG₁ (Zymed) at 4° C. for 1 hr. Thecells were then recovered, washed and lysed in a lysis buffer (20 mMTris.HCl, pH 7.4, 140 mM NaCl, 1% digitonin, 1 mM sodium vanadate, 5μg/ml aprotinin and 1 μg/ml leupeptin) on ice for 15 min. The 4-1BB and53A2 complexes were precipitated by rec-protein G Sepharose-4B (Zymed).The immunoprecipitates were run on 10% SDS-polyacrylamide gel andexposed to X-ray film after soaking the gel in EN³HANCE (NEN).

[0204] Flow Cytometry

[0205] Cells (0.5×10⁶/sample) were incubated with in RPMI 1640, 1% BSA,and 0.1% sodium azide with purified 53A2 on ice for 30 min. Cells werewashed three times in RPMI 1640 containing 5% FBS. The cells were thenincubated on ice for 30 min with fluorescein Isotiliocyanate(FITC)-conjugated goat anti-rat immunoglobulin. Some samples receivedFITC-conjugated goat anti-rat Ig only. The cells were washed again withphosphate buffered saline (pH 7.4) containing 10% bovine serum albulnin.Flow cytometry was performed using an EPICS 753 (Coulter)Florescence-activated cell sorter.

[0206] Northern Blot Analysis

[0207] Thymocytes or spleen T cells were stimulated with anti-CD3 MAb(clone 145-2C11), ionomycin PMA (Calbiochem, La Jolla, Calif.), 10 ng/mePMA (Sigma, St. Louis, Mo.), or ionomycin plus PMA. Individual wells of96-well flat bottomed culture plates or T25 Flasks (Costar, Cambridge,Mass.) were coated with 10 micrograms/ml anti-CD3 in PBS, pH 7.0 for 3hours at 37° C. and the washed thress times with PBS. For RNAextraction, 2 to 3×10⁷ cells at 5×10⁶/ml were stimulated in completemedium and harvested at the indicated time points. Total RNA wasextraceted by the guanidinium-phenol extraction procedure (102). The RNAwas fractionated on a 1.4% formaldehyde denaturing agarose gel,transferred to a Gene-Screen Plus membrane, and hybridized 32p labeledprobes.

[0208] Results

[0209] In order to study the 4-1BB protein (4-1BBP), polyclonalantibodies were raised against oligopeptides representing five differentportions of the predicted 4-1BBP. To aid in proving that the putativeantisera contain antibodies which uniquely, recognize the 4-1BBP, anexpression plasmid was contructed contining the putative extracellularportion of 4-1BB cDNA. The trancated cDNA was expressed in a baculoviralexpression system. The antibodies were tested to see if any recognizedthe recombinant soluble 4-1BBP (rs-4-1BBP) in the Sf-21 cell culturemedium. Two antisera among five tested recognized a protein found onlyin the supernatant of 4-1BBS infected cells. One anti-oligopeptideantiserum, anti-4-1BB-0, stained the band at a higher dilution (1:1600)than did the other one, anti-4-1BB-11. Rs-4-1BBP was purified from Sf-21cell culture medium.

[0210] As shown in FIG. 21 (arrows), anti-rs-4-1BBP-reacting bands weredetected of approximatley 18, 20, and 23 kDa (FIG. 21: Lane 1, Coomassieblue staining; lane 2, anti-4-1BB-0 antibody staining, and lane 3,molecular size marker). Partial amino acid sequences of each of thethree bands were determined. The sequences of the 3 bands were identicaland were Val-Gln-Asn-Ser-X-Asp. The amino acid sequence at positions 1,2, 3, 4 and 6 was identical to that of the mature 4-1BBP predicted fromthe cDNA sequence. Amino acid at position 5 which was supposed to be Cyswas not determined. Also, rs-4-1BBP could be immunoprecipitated by 53A2,an anti-4-1BB monoclonal antibody. Production of the three differentsizes of rs-4-1BBP is most likely due to the differences inglycosylation. These results indicate that the deduced amino acidsequence and assignment of signal sequence were correct. When thepotential transmembrane domain was removed from the complete 4-1BBmolecule, the protein was secreted, which is consistent with the fulllength 4-1BBP being associated with the cellular membrane as predictedby the primary structure.

[0211] Biochemical Properties of 4-1BB

[0212] Full length 4-1BB cDNA (4-1BBL) was expressed, in Sf-21 cellsusing recombinant baculoviruses containing 4-1BBL. The cell lysates werefractionated on a 10% SDS polyacrylamide gel and transferred toImmobilon-P. The blot was incubated with 53A2, an anti-4-1BB monoclonalantibody, and stained with goat-anti-rat IgG-conjugated alkalinephosphatase.

[0213]FIG. 22 shows recombinant 4-1BB protein. Full length 4-1BB cDNA(4-1BBL) was expressed in insect cells (sf 21) using recombinantbaculoviruses containing 4-1BBL. The cell lysates were fractionated on a10% SDS polyacrylamide gel, transferred to Immobilon-p and stained with53A2, an anti-4-1BB mAb. The monomer of 4-1BB in reducing conditionsconsists of three major species whose sizes are 29, 30 and 31 kDa (lane2). Non-reducing conditions (lane 4-) produced dimerized 4-1BB whosesizes are 49 to 60 kDa. Some of 4-1BB molecules are monomerized in lane4, showing both monomers and dimers. Lane 1, uninfected Sf 21 celllysate; lane 2, 4-IBBL-infected Sf2I cell lysate; lane 3,MIP-1β-infected Sf2l cell lysate (unrelated to 4-1BBL); lane 4,4-1BBL-infected Sf 21 cell lysate.

[0214] Under reducing conditions, the 4-1BB monomer consisted of threemajor species corresponding to 29, 30 and 31 kDa (FIG. 22, lane 2).Under non-reducing conditions, 4-1BB existed as a monomer as well as adimer of 49 to 60 kDa. The heterogeneity in size may be due to differentamounts of glycosylation as seen in rs-4-1BBP (FIG. 21).

[0215]FIG. 23 shows an immunoprecipitation of cell surface 4-1BB proteinsynthesized by T lymphocytes. T cell (F1 clone) surface 4-1BB proteinwas immunoprecipitated with anti-4-1BB mAb 53A2 (lanes 2 and 4) or withrat IgG¹ control mAb (lanes 1 and 3). Lanes 1 and 2 were run underreducing conditions and lanes 3 and 4 were run under nonreducingconditions. Monomeric form of 4-1BB protein is indicated by allarrowhead with number 1. The dimeric and tetrameric forms are indicatedby the arrowheads with numbers 2 and 3, respectively.

[0216] 4-1BBP expressed on T lylnpliocytes was immunoprecipitated withthe mAb 53A2. A CD4⁺ T cell clone F1 was labeled with [³⁵S]cysteine. ThemAb 53A2 (FIG. 23, lanes 2 and 4) or rat IgG¹ control (FIG. 23, lanes 1and 3) antibodies was first incubated with F1 cells to bind to surface4-1BBP. The cells were subsequently washed to remove unbound antibody,lysed, and immunoprecipitated wihl protein G-conjugated Sepharose beads.Under reducing conditions, 53A2 immunoprecipltated a 30-kDa protein(FIG. 23, arrow 1) and under non-reducing conditions, 53A2immunoprecipitated 30-kDa (FIG. 23, arrow 1), 55-kDa (FIG. 23, arrow 2)and 110-kDa (FIG. 23, arrow 3) proteins. These three bands may representmonomer, dimer and tetramer species, respectively. Whether all thesethree forms of 4-1BBP exist in the T cell membrane and are in a dynamicequilibrium is not known. The most abundant form appears to be thedimeric 4-1BB.

[0217] Expression of 4-1BBP in Splenic T Cells and Thymocytes

[0218] Splenic T cells were isolated by a nylon wool column and apercoll gradient. The T cells were stimulated with 10 μg/ml ofimmobilized anti-CD3 mAb. T cells were harvested at various time pointsand tested for 4-1BB mRNA expression by Northern blot analysis, As shownin FIG. 24, 4-1BB mRNA was detected by 3 hrs of stimulation. Northernanalysis also indicated that 4-1BB mRNA was not expressed inLPS-activated B cell blasts or in A20 cells, a B cell lymphoma (data notshown).

[0219]FIGS. 24a and 24B show that 4-1BB mRNA expression in inducedmurine splenic T cells by anti-CD3-activation. Splenic T cells wereisolated by a nylon wool column and a percoll gradient (cells wereisolated at the 60-70% interface) and stimulated over time with 10,μg/ml of immobilzed anti-CD3 (145-2C11) in the presence of accessorycells. Cyltures were harvested at indicated time points and monitoredfor 4-1BB expression by Northern analysis. Each lane contains 20 μg oftotal RNA. Northern blot was hybridized with a 4-1BB cDNA probe (23a)and the gel was stained with ethidium bromide prior to transfer as acontrol for equal loading of each lane.

[0220] Thymocytes were stimulated with ionomycin, PMA or ionomycin plusPMA and tested for 4-1BB mRNA expression. As shown in FIG. 25, 4-1BBmRNA was inducible by ionomycin or PMA. Both ionomycin and PMA, however,were required for optional expression of 4-1BB mRNA, indicating that4-1BB mRNA expression requires protein kinase C activation and increasesin intracellullar Ca²⁺.

[0221]FIG. 25 shows that optimal induction of 4-1BB mRNA requires bothProtein Kinase C activation and increases in intracellular Ca²⁺.Thymocyte culture were stimulated with medium alone (lane 1), 1 μMionomycin (1) (lane 2), 10 ng/ml TPA (T) (lane 3), T+1 (lane 4) for 6hours and monitored for 4-1BB mRNA expression by Northern analysis. RNAwas also isolated from CTLL-R8 cells and monitored for 4-1BB mRNA byNorthern analysis (lane 5). Each lane contains 10 μg of total RNA. Blotwas hybridized sequentially with a 4-1BB cDNA probe (24a) and with aCHOB probe (24b) as a control for equal loading in each lane.

[0222] The induction of 4-1BB mRNA was blocked by cycloheximidetreatment, indicating that 4-1BB mRNA expression requires new proteinsynthesis (FIG. 26). Therefore, 4-1BB is classified as a member of theearly gene family (58).

[0223]FIG. 26 shows transaction factors necessary for the induction of4-1BB mRNA expression do not pre-exist in the resting T cell.Cycloheximide (CHX), a protein synthesis inhibitor, blocks the inductionof 4-1BB mRNA in TPA alld ionomycin-activated thymocytes. Thymocytescultures were stimulated with medium alone (lane 1), 20 μg/ml CHX (lane2), T+1 (lane 3), or CHX +T+1 (lane 4) for 6 hours and monitored for4-1BB mRNA expression by Northern blot analysis. Each lane contains 10μg of total RNA. Blot was hybridized sequentially with a 4-1BB-cDNAprobe (24a) and with a CHOB cDNA probe (4b) as a control for equalloading in each lane.

[0224] 4-1BBP was expressed on the surface of activated thymocytes,splenic T cells, CD4⁺ and CD8⁺ T cells. As shown in FIG. 27, 4-1BBP wasinducible when the thymocytes were treated with ionomycin plus PMA (FIG.27a, peak 3) and when the splenic T cells were treated with anti-CD3 mAb(FIG. 27b, peak 3). The 4-1BBP was also induced in the CD4⁺ T cellclone, F1, by anti-CD3 mAb (FIG. 27c, peak 3) and was expressedconstitutively in CTLL-R8 cells (FIG. 27d, peak 2).

[0225]FIGS. 27a-d show that 4-1BB is expressed on the cell surface ofactivated thymocytes, splenic T cells, CD4⁺ and CD8⁺ T cells. AThymocytes were stained with FITC-conjugated-goat-anti-rat IgG(anti-IgG-FITC) alone (peak 1) or with anti-4-1BB mAb plus anti-IgG-FITCon control thymocytes (peak 2) or thymocytes stimlulated with T+1 for 12hrs (peak 3). B, Splenic T cells were stained with anti-IgG-F1TC alone(peak 1) or with anti-4-1BB mAb plus anti-IgG-FITC on resting T cells(peak 2) or T cells stimulated with immobilized anti-CD3 for 12 hours(peak 3), C, A CD4⁺ T cell clone, F1, was stained with anti-TgG-FITCalone (peak 1) or with anti-4-1BB mAb plus anti-IgG-F1TC on control F1cells (peak 2) or F1 cells stimulated with immobilized anti-CD3 for 24hrs (peak 3). D, A CD8⁺ T cell line, CTLL-R8, was stained withanti-IgG-FITC alone (peak 1) or with anti-4-1BB mAb plus anti-IgG-FTTC(peak 2).

[0226] Effect of the Anti-41BB mAb, 53A2, on anti-CD3-Stimulated T CellProliferation

[0227] To determine whether biochemical signals delivered through 4-1BBmay contribute to T cell activation, the anti-4-1BB mAb, 53A2, waspotentially used to mimic ligand billdilig to cell surface 4-1BB.Purified resting splenic T cells were stimulated with 10 μg/mlimmobilized anti-CD3 in the absence or presence of 53A2.

[0228]FIG. 28 shows anti-4-1BB mAb, 53A2, enhances the prolifeation ofanti-CD3-activated splenic T cells. Purified splnic T cells (5×10⁴cells/well) were stimulated in triplicate with 10 micrograms/mlimmobilized anti-CD3 alone or in the presence of 10 micrograms/ml 53A2or 10 micrograms/ml rat IgG₁ as a control. cultrues were incubated forthe indicated times and plused for 12 to 14 hours with 1μCi[³H]thymidine. The cultures were harvested and [³H]thymidineincorporation was measured by liquid scintillation counting.

[0229] In FIG. 28, on days 3-5 of activation, all enhancement of ³Hthymidine incorporation was observed in T cells stimulated with anti-CD3in the presence of 53A2. Enhancement of proliferation was not presentwhen T cells were stimulated with anti-CD3 in the presence of a ratisotype-matched control antibody. In data not shown, the 4-1BBanti-peptide antiserum, 4-1BB-0, had no effect on anti-CD3-mediated Tcell proliferation. TABLE 4 Day 3 Day 4 Day 5 [³H]TdrIncorporation—Experiment I Control 320 = 57  715 = 74  790 = 117 53A2(10 μg/ml) 1103 = 60  2268 = 232  3323 = 717  IgG₁ (10 μg/ml) 710 = 95 1501 = 165  1553 = 196  αCD3 (10 μg/ml) 3164 = 714  11217 = 735  17380 =420  αCD3 + 53A2 44155 = 665  35796 = 370  60443 = 803  (10 μg/ml)αCD3 + IgG¹ 7945 = 1217 10404 = 1999  17843 = 2490  (10 μg/ml) Foldincrease αCD3 vs 9.6 x 3.0 x 3.5 x αCD3 53A2 [³H]TdrIncorporation—Experiment II Control 336 = 185 284 = 35  120 = 26  53A2(10 μg/ml) 387 = 69  326 = 36  166 = 71  IgG₁ (10 μg/ml) 381 = 224 302 =87  229 = 85  αCD3 (10 μg/ml) 16999 = 971  15823 = 209  6877 = 1176αCD3 + 53A2 30570 = 1250  29875 = 343  10668 = 1165  (10 μg/ml) αCD3 +IgG¹ 17638 = 3274  15365 = 233  6238 = 110  (10 μg/ml) Fold increaseαCD3 vs 1.8 x 1.9 x 1.6 x αCD3 53A2

[0230] In other experiments, the actual degree of enhancement rangedfrom an approximately 2-10fold increase in [³H]thymidine incorporationin culturses stimulated with anti-CD3 alone (Table 4). Differences inactual enhancement of ^([3)H] thymidine incorporation, could be due tovariability in the nunber or metabolic status of accessory cells in thecultures. For example, the highest-fold increase in T cells stimulatedwith anti-CD3 in the presence of 53A2 generally occurred when theproliferative effects of anti-CD3 were miniml (Table 4). It will benecessary to obtain T cell populations of higher purity to address thisissue. These data, however, conclusively show that 4-1BB-mediatedsignals can contribute to T cell prolferation.

[0231] Discussion

[0232] These experiments represent a further characterization of 4-1BBand show that 4-1BB may function as a cell surface receptor capable oftransmitting biochemical signals during T cell activation.

[0233] The major species of 4-1BB on the cell surface appears to be a55-kDa dimer. 4-1BB also appears to exist as a 30-kDa monomer andpossibly as a 110-kDa tetramer. Since these 4-1BB species wereimmunoprecipitated from a homogenous population of cells (T cell cloneF1), all forms potentially co-exist on each cell. A comparison ofpeptide digests from the 4-1BB monomer and dimer will be needed todetermine whether 4-1BB exists as a homodimer on the cell surface. Avariety of cell surface receptors such as the insulin receptor (59), theB cell surface immunoglobulin receptor (60), the T cell Ag receptro(61), the CD28 costimulatory receptor (62), and the CD27 T cell antigen(63) are composed of disulfide-bonded subunits. Receptor dimerizationmay be required for ligand binding and subsequent biochemical signaling.

[0234] 4-1BB is not expressed on resting T cells but is inducible byactivators which deliver a complete growth stimulus to the T cell. Thecombination of PMA and ionomycin is capable of mimicking those signalsrequired for T cell proliferation. Although PMA or ionomycin aloneinduced 4-1BB mRNA, the combination of PMA and ionomycin resulted inoptimal 4-1BB expression. Furthermore, the expression of 4-1BB was nottransient. When purified splenic T cells were stimulated withimmobilized anti-CD3, 4-1BB mRNA was expressed and this expression wasmaintained for up to 96 hrs poststimulation. Cell cycle analysis will berequired to confirm that 4-1BB is expressed throughout cell cycleprogression.

[0235] 4-1BB is structurally related to members of the nerve growthfactor receptor super-family. Although these receptors possessstructurally similar ligand-binding properties (cysteine-rich regions),the cytoplasmic domains of these proteins are nonconserved which couldallow for diversity in transmembrane signaling. Some members of thisfamily are involved in the T or B cell activation process. There are invitro functional data on the OX-40, CD4O and CD27 antigens. Antibodiesagainst the OX-40 angment the T cell response in a mixed lymphocytereaction (47) and antibodies against CD4O enhance B-cell proliferationin the presence of a coactivator, such as PMA or CD2O antibodies, andsynergize with IL-4 in vitro to induce B-cell differentiation and togenerate long-term normal B cell lines (64). One inonoclonal antibody,anti-1A4, which recognizes an epitope on the CD27 molecule inhibitedcalcium mobilization, IL-2 secretion, helper T cell function, and T cellproliferation. On the other hand, CLB-CD27/1, another anti-CD27 mAbenhanced proliferation of hunan T cells stimulated with PHA or anti-CD3mAb (63). These results indicate that the CD27 molecule plays animportant role in T cell activation. Except for TNFRs, NCFR and CD4O,the ligands or cell surface molecules to which the members of thesuperfamily bind are not yet identified. Identification andcharacterization of the ligands to which the receptors bind will behelpful in better defining the physiologic role of 4-1BB.

[0236] To ascertain whether cell surface 4-1BB could contribute to Tcell activation, the anti-4-1BB 53A2 was used as an agonist to 4-1BB.These data suggested that 4-1BB does in fact have the potential tofunction as an accessory signaling molecule during T cell activation andproliferation. The addition of soluble 53A2 to purified splenic T cellsstimulated with immobilized anti-CD3 resulted in an amplification of ³Hthymidine incorporation compared to T cells stimulated with anti-CD3alone. This pattern of enhancement ranged from 2- to 10-fold in threeindependent experiments.

[0237] In the original two signal model of Bretschier and Cohn, theyproposed that signal 1, the occupancy of the T cell antigen receptor(TCR), resulted in inactivation f the T cell in the absence of signal 2,which is provided by accessory cells. This as since been confirmed by avariety of studies (65). The identification of the accessory cell CD28as a potent costimulatory receptor on T cells was a significantcontribution in beginning to charactize the accessory signal(s) requiredfor optimal T cell proliferation (66). It is possible that other cellsurface molecules may contribute to these costimulatory activationrequirements (67).

[0238] The biochemical signals delivered through 4-1BB are notcompletely known. One possibility considered was the observation that4-1BB contains a putative p56^(lck) tyrosine kinase binding doimain inits cytoplasmic tail. It was later determined that p56^(lck) tyrosinasekinase binds to 4-1BB. It will also be worthwhile to determine if4-1BB-mediated signaling can regullate genes such as IL=2 and IL-2receptor, whose expression is required for T cell activation andsubsequent proliferation.

[0239] Although the precise functions of member of the NGFR familyappear to be diverse, an emerging theme is one in which these moleculesmay contribute in various ways to a maintenance of responsiveness orviability of the particular cell type in which they are expressed. Forinstance, NGF is absolutely required for viability of neurons in vitroand in vivo (68). The crosslinking of CD40 by soluble antiCD4-0monoclonal antibody blocks germinal center centrocytes from undergoingapoptosis in vitro (69). Signals delivered throug CD4O may also aid inmaintenance of responsiveness to differeniation factors. The ligation ofCD4O with antl-CD4O F(ab′)₂ fragments in the presence of IL-4 inducedlarge increases IgE synthesis (70). Also, anti-CD40 activated naive Bcells treated with IL-10 and transforming growth factor-β becamecommitted to IgA secretion (71).

[0240] In addition to sharing the molecular characteristics with theNGFR superfamily, it was noted that the 4-1BB contained a putative zincfinger structure of the yeast elF-2 μl protein (110). FIG. 17 shows acomparision of the 4-1BP amino acid sequence with the amino acidsequence in sina seven in absentia of Drosophila and DG17 ofDictyostelium. The amino acids that are share are boxed. Numberrepresent the positions of the left-most residues relative to theN-terminus. Gaps (−) are introduced to allow for maximal alignment.4-1BB also shares a conserved region with the sina seven in absentia ofDrosophila, which is required for correct photoreceptor cell development(108). That particular region is also similar to the protein product ofthe DG17 gene of Dictyostelium, whose expression is specificaily inducedduring aggregation by cAMP (FIG. 17) (109).

[0241] This region forms the pattern of C—X₂—C—X₉—C—X₃—H—X₃—C—X—C; andthe cysteines and histidine are conserved in a similar space in 4-1BB,sina, and DG17 proteins. Ten of 24 amino acids between the 4-1BB andsina proteins are identical, and 3 of 24, are conservative substitutes.The conserved pattern suggests that these amino acids are functionallyimportant. The sina protein is localized in the nucleus, suggesting thatit has a regulatory function in cells. The fact that the amino acidsequence of 4-1BB contains features like a zinc finger motif, a nuclearprotein, and a receptor domain suggests that 4-1BB may play diverseroles during cellular proliferation and differentiation.

[0242] A T Cell Antigen 4-1BB Associates With the Protein TyrosineKinase p56^(lck)

[0243] 4-1BB is a 30 kD inducible T-cell antigen, and is expressedpredominantly as a 55 K dimer on both CD4+ and CD8⁺ T lymphocytes Thecytoplasmic tail of 4-1BB contains the sequence, Cys-Arg-Cys-Pro, whichis simijar to the sequence Cys-X-Cys-Pro, that mediates the binding ofthe CD4 and CD8 molecules to p56^(lck) a protein tyrosine kinase^(2,3).An anti-4-1BB monoclonal antibody (53A2 mAb) was used to determinewhether 4-1BB may associate with p56^(lck). The 53A2 mAb specificallyrecognized 4-1BB on a CD8⁺ T-cell line, CTLL-2, and coimmunoprecipitateda 56 K protein along with 4-1BB. Peptide mapping indicated that the 56 Kphosphoprotein was identical to p56^(lck). The coimmunoprecipitation ofp56^(lck) with 4-1BB also occurred in nonlymphoid cells such as insect(Sf-21) and HeLa cells when the two recombinant proteins werecoexpressed. Analysis of mutant p56^(lck) recombinant proteins showedthat two cysteine residues, critical for p56^(lck)-CD4 (or CD8) complexformation, are also required for the P56^(lck)-4-BB interaction. Thesestudies establish that 4-1BB physically associates with p56^(lck).

[0244] The T-cell activation marker, 4-1BB, is related structurally tomembers of the nerve growth factor receptor (NGFR) super-family, whichare characterized by the presence of three to six patterns of acysteine-rich motif in their extracellular domains. The members of NGFRsuper-family also contain hinge-like regions abutting theirtransmembrane domains with a high proportion of serine/threonine, likelyto be glycosylated with O-linked carbohydrates. The cytoplasmic domainsof these receptors show no sequence similarities; therefore, diversesignaling pathways may be operative. 4-1BB expression is induced onCD4-⁺ or CD8⁺T-cell surface by various T-cell mitogens or crosslinkingof T-cell receptors (TCR). The calculated molecular weight of 4-1BBpredicted from the cDNA nucleotide sequence is 25, 167 (233 aminoacids). 4-1BB, however, migrates on a SDS polyacrylamide gel at a sizeof 28 to 30 K, suggesting its possible posttranslational modification.

[0245] An interesting feature of 4-1BB is its cytoplasmic domaincontaining a putative binding site for the T-cell-specific proteintyrosine kinase, p56^(lck) a member of the tyrosine kinase family, iscoupled to CD4-, CD8 (72,93), 3 PLC-γ (73) and the IL-2 receptor (74).Recently, other T cell antigens; e.g., CD5(75) and CD2(76) have beenlisted as proteins associated with p56^(lck) in human and rat cells,respectively. The consensus sequence for the p56 ^(lck) binding site is—C—X—C—P— in the CD4 and CD8 molecues (72,93). p56^(lck) is expressed athigh levels in both developing and mature T cells and its activity ismodulated during T-cell activation (77).

[0246] The role of 4-1BB during T-cell effector function is not entirelyclear. A rat anti-mouse monoclonal antibody to 4-1BB, 53A2 (IgG,) asdisclosed herein which specifically recognizes an epitope on theextracellular domain of native and recombinant 4-1BB.

[0247] It has shown that 53A2 enhanced the anti-CD3-mediatedproliferation of splenic T-cells, suggesting an involvement of 4-1BBduring T-cell activation. In this study, 4-1BB ws shown to physicallyassociates with p56^(lck). It is now reported that in T-cells, 4-1BB iscomplexed with a kinase identified as p56^(lck). 4-1BB and p56^(lck)recombinant proteins are also associated when they are coexpressed innonlymphoid cells. The interaction between 4-1BB and p56^(lck) requirestwo critical cysteines of p56^(lck) which are also essential for bindingto CD4 or CD8 molecules in T cells.

[0248]FIGS. 29a-c show specificity of 53A2 mAb to native and recombinant4-1BB. FIG. 29a shows the expression of 4-1BB on CTLL-2 cells. For FACSanalysis, CTLL-2 cells were cultured for 16 hrs in the presence of 100u/ml IL-2 without or with anti-CD3 (145, 2C11) (1 ug/ml)(134). Cellswere incubated with 53A2 (10 μg/ml) in 2% fetal calf serum (FCS)containing RPMI-IM-0 at 4-° C. for 1 hr followed by FITC-goat anti-ratIg antibodies. Peak I represents the background fluorescence from CTLL-2cells treated with F1TC-labeled goat anti-rat Ig alone. Peaks 2 and 3indicate staining of cells cultured in the absence or presence ofantl-CD3, respectively. FIG. 29b shows the expression of recombinant4-1BB on insect cells (Sf-21). Insect cells infected with wild-typebaculoviruses (FIG. 290b, peak 1), 4-1BB recombinant baculoviruses for 1day (FIG. 29b, peak 2) and for 2 days (FIG. 29b, peak 3) were used forFACS analysis of 4-1BB with 53A2. FIG. 29c shows the immunoprecipitationanalysis; 2×10⁷ CTLL-2 cells were metabolically labeled overnight with[35S]-cysteine (0.1 mCi/ml) in cysteine-free RPM1-1640, 10% dialyed FCS,2 mM glutamine, 1 mM sodium pyruvate, 5×1O-5M 2-mercaptoethanol and 100u/ml IL-2. Cells were lysed in 100 pl of 20 mM Tris-HCI, pH 7.4, 140 mMNaCl, 1% digitonin, 1 mM sodium orthovanadate, and 10 pg/ml aprotinin onice for 15 min. Following microcentrlfugation at 10,000×g for 10 min toremove cell debris, the cellular lysate was incubated withiso-type-matched rat IgG₁ control (Zymed Lab. Inc.) (FIG. 29c, lane 1)or with anti-4-1BB mAb 53A2 (FIG. 29c, lane 2) at 4° C. for 1 hr. Theimmune complexes were recovered using 50 μl rec-protein G Sepharose 4Band washed with the lysis buffer, phosphate buffer containing 0.5M NaCl,then finally with PBS, and rEn on a 10% SDS polyacryamide gel underreducing conditions. The gel was processed with EN³HANCE (NEN) forfluorography and exposed to X-ray film (Kodak XAR-5) for two days. Thearrow indicates the 4-1BB proteins.

[0249] FACS analyses indicated that 53A2 specifically recognized 4-1BBexpressed on cell surface of Sf-21 cells infected with 4-1BB-expressingbaculoviruses. Sf-21 cells were infected with wild-type as a control(FIG. 29, peak 1) and 4-1BB recombinant baculoviruses for 1 (FIG. 29,peak 2) or 2 (FIG. 29, peak 3) days and assayed for cell surfaceexpression of 4-1BB by FACS with 53A2 and subsequent FITC conjugatesecondary antibody. 4-1BB was expressed on the stlrface of CTLL-2 cells(FIG. 29, peak 2) but at higher levels on CTLL-2 cells stimulated withanti-CD3 (145-2C11) for 16 hrs (FIG. 29, peak 3).

[0250] The 4-1BB antigen was immunoprecipitated with 53A2 from a lysateprepared from a metabolically [³⁵S] cysteine-labeled CTLL-2 (FIG. 29b).4-1BB migrated on a 10% SDS polyacrylamide gel at approxirnately M_(r)of 28 K under reducing conditions. When the immunoprecipitate was rununder non-reducing collditions, an additional form of 4-1BB was detectedat M_(r) of 55 K, suggesting the presence of a 4-1BB dimer in CTLL-2cells (data not shown). Depending on lysis and washing conditions, weoften observed extra bands at about M, of 45 K in 53A2-immunoprecipitesof CTLL-2 cells.

[0251] The physical association between 4-1BB and p56^(lck) was examinedby a coimmunoprecipitation assay with 53A2 followed by an in vitrokinase-labeling reaction in the presence of [γ-³²P] ATP. This in vitrokinase assay provides greater sensitivity in monitoring the appearanceof phosphoproteins in the immune complexes, and takes advantage of thefact that p56^(lck) undergoes autophosphorylation, assuming thatsubstrates would be phosphorylated by any associated kinase activity inthe immune complex. CTLL-2 cells were lysed with 1% digitonin, and thelysate was immunoprecipitated with isotype-matched rat IgG19 53A2 oranti-p56^(lck) serum (72). The immune complexes were incubated with[γ-³²P] ATP and ³²P-labeled proteins were detected by autoradiography.

[0252]FIGS. 30a and 30 b show the identification of thecoimmunoprecipitated proteins. FIG. 30a shows the communoprecipitationof 4-1BB and p56^(lck) A CTLL-2 cell lysate was incubated withisotype-matched rat IgG, (lane 1), anti-4-1BB mAb 53A2 (lane 2) oranti-56^(lck) (lane 3). The antip56^(lck) is a polyclonal rabbit serumraised against a synthetic peptide corresponding to amino acids 39-64 ofthe murine p56^(lck) protein (72). The immune complexes wereprecipitated as before, and subjected to the in vitro kinase reactionwith 30 μCi [γ-³²P] ATP in 25 pl 25 mM HEPES, pH 7.4, 10 mM MnCI, and0.1% NP-4-0 at 30° C. for 15 min. The reaction was terminated by adding6 μl of 5×SDS sample buffer and boiling for 5 min. The ³²P-labeledimmune complexes were run on a 10% SDS polyacrylamide gel, transferredto Immobilon-P and exposed to S-ray film. The arrow indicates the 56 Kphosphorylated bands (FIG. 30a). FIG. 30b shows V8 protease peptidemapping. ³²P phosphorylated proteins that were immunoprecipitated with53A2 (FIG. 30b, lane 1) or with anti-p56^(lck) (FIG. 30b, lane 3) wererecovered and partially digested with S. aureus V8 protease (135). V8protease-digests of lanes 1 and 3 correspond to the samples in lanes 2and 4, respectively. The samples were resolved on a 15% SDSpolyacrylamide gel under reducing conditions and exposed to X-ray film.Small arrows indicate the V8 protease digestion products in lanes 2 and4 and the large arrow indicates the undigested phosphoproteins in lanes1 and 3.

[0253] 53A2 (FIG. 30, lane 2) coimmunoprecipitated a phosphoprotein withan M_(r) of −56 K that migrated similarly to p56^(lck)immunoprecipitated by anti-p56^(lck) serum (FIG. 30a, lane 3). Theimmunoprecipitate from isotype-matched rat IgG, however, did not showany detectable phosphoprotein (FIG. 30, lane 1). To confirm that the4-1BB-associated molecule was p56^(lck), both phosphoproteins from 53A2or anti-p56^(lck) immunoprecipitations by digestion Niith S. u˜ir.us V8protease (FIG. 30b, lanes 2 and 4, respectively). Lanes 1 and 3represent ³²P-immunoprecipitates using 53A2 and anti-p56^(lck)respectively. The digestion patterns of phosphoproteins generated fromcoimmunoprecipitates of 53A2 were identical to those fromanti-p56^(lck). These results suggest that 4-1BB associates withp56^(lck) in CTLL-2 cells.

[0254]FIGS. 31a-c show an analysis of the association of 4-1BB andp56^(lck) in a baculoviral expression system. FIGS. 31a and 31 b showand immunoblot of 4-1BB and p56^(lck) Sf-21 insectcellswere infectedwith 4-1BB-, p56^(lck)-expressing recombinant baculoviruses orcoinfected with 4-1BB and p56^(lck)-expressing recombinantbaculoviruses. Total lysates from Sf-21 cells infected with theserecombinant baculoviruses were blotted and probed with rabbit anti-4-1BBand rabbit anti-p56^(lck) (FIGS. 31a and 31 b, respectively). Antigenswere visualized with alkline phosphatase-conjugated secondary artibodiesand chromogenic substrates, NBT and BCIP. Anti-4-1BB polyclonal rabbitserum was raised against the oligopeptide, CRPGQELTKQG, whichcorresponds to amino acids 82 to 92 of mature 4-1BB. FIG. 31c shows animmune complex kinase assay of p56^(lck). These Sf-21 cell lysates werealso incubated with isotype-matched rat IgG₁ (FIG. 31c, lane 1), 53A2(FIG. 31c, lane 2) or anti-p56^(lck) (FIG. 31c, lane 3). The immunecomplexes were precipitated, subjected to the in vitro kinase reactionwith [γ-³²P] ATP and run on a 10% SDS-polyacrylamide gel as described inFIG. 30. The arrow indicates the autophosphorylated p56^(lck) proteins.

[0255] Sf-21, an insect cell line derived from Spadoptera fragiperda wasgrown at 27° C. in a synthetic serum-free medium, Excell 400 (JRHBioscience) contining antibiotics. To generate recombinant 4-1BB orp56^(lck) expressing baculoviruses, 4-1BB and murine p56^(lck) cDNA'swere cloned into PEV 55 baculoviral transfer vector (136) andTransferred to the Autographa californica nulear polyhedrosis virus(AcNPV) genome as described elsewhere (53). 4-1BB or p56^(lck) cDNAcontaining baculoviruses were plaque purified and were grown in Sf-21cells. 4-1BB or p56^(lck) was expressed by infecting the Sf-21 cellswith the recombinant viruses at a multiplicity of infection of 5 for 3days. The infected cells were harvested, washed and lysed in the lysisbuffer containing 1% digitonin for immunoprecipitation analysis.

[0256] The association of 4-1BB with p56^(lck) as examined innonlymphoid cells to confirm the specificity of the 4-1BB-p56^(lck)complex, and to rule out the possibility that other T-cell-specificfactors were involved in this interaction. To demonstrate the directphysical association of 4-1BB with p56^(lck,) 4-1BB was coexpressed with!nurine p56^(lck) in insect (Sf-21) cells infected with recombinantbaculoviruses (FIG. 31). An immunoblotting analysis was performed toensure that recombinant proteins 4-1BB (FIG. 31a) and p56^(lck) (FIG.31b) were expressed in the appropriate infections. We used polyclonalrabbit antiserum raised against an 11 amino acid oligopeptide(cysteine-82 to g)ycine-92 in 4-1BB) was used for detection of 4-1BB,which allowed for enhanced detection of transblotted 4-1BB. Recombinant4-1BB protein detected by immunoblotting appeared as multiple bands,presumably as a result of posttranslational modifications. Lysates fromSf-21 cells infected with 4-1BB-,p56^(lck)-recombinant baculoviruses orcoinfected with both recombinant baculoviruses (FIG. 31) were subjectedto immunoprecipitation with control rat IgG, (lane 1), 53A2 (lane 2) oranti-p56^(lck) (lane 3) followed by in vitro phosphorylation in thepresence of [γ-³²P] ATP. Although 4-1BB was expressed in Sf-21 cellsinfected with 4-1BB recombinant baculoviruses (FIG. 31), none of theantibodies mentioned above immunoprecipitated a tyrosine-phosphoproteinfrom this lysate (FIG. 31, infection-4-1BB). This indicated that noendogenous tyrosine kinase capable of phosphorylating 4-1BB or p56^(lck)existed in Sf-21 cells. As expected, only anti-p56^(lck)imnlunoprecipi˜ted p56^(lck)-phosphoprotein from a lysate prepared fromcells infected with baculovirEses expressing p56^(lck) (FIG. 31,infection-LCK, lane 3). Likewise, 53A2 and anti-p56^(lck) but notcontrol rat IgG₁, immunoprecipitated the p56^(lck) phosphoprotein from aSf-21 lysate containing both p56^(lck) and 4-1BB (FIG. 31,infection-4-1BB+LCK, lanes 2 and 3). These results from nonlymphoidcells demonstrated that 4-1BB specifically associated with p56^(lck) andexcluded the possibility that other T-cell factors were required for theinteraction.

[0257] The interaction between CD4 or CD8 and p56^(lck) requires aspecific binding site on each molecule^(2,3). The N-terminal region ofp56^(lck) interacts noncovalently with the cytoplasmic domains of CD4and CD8 via pairs of cysteine residues in each moleculez, Therefore, itwas determined whether the same cvsteines of p56^(lck) (cysteine 20 and23) are required for the association with 4-1BB. p56^(lck) constructscontaining sequences encoding muatted p56^(lck) proteins (construct C₁,cys 20-ser; construct C₂, cys 23-ser)² were employed in the followingexperiments. The capability of the cysteine-mutant p56^(lck) proteins toassociate with 4-1BB was tested by the coimmunoprecipitation assay. Avaccinia virus expression system was utilized in which the T7 RNApolymerase-expressing vaccinia virus allows transfected genes under thecontrol of the T7 promoter to be expressed in HeLa cells². High levelsof coexpression have been achieved previously with this system tofacilitate interactions between CD4 and p56^(lck). To ensure consistentexpression of 4-1BB, the transfected HeLa cells were briefly labeledwith [³⁵S] cysteine before harvest. 4-1BB expression was monitored byimmunoprecipitation of ³⁵S-labeled 4-1BB with 53A2.

[0258]FIGS. 32a-c show an analysis of the association of 4-1BB andp56^(lck) HeLa cells. FIG. 32a shows an immunoblot of p56^(lck) HeLacells were transfected with cDNAs encoding the indicated proteins andmetabolically labeled with [³⁵S] cysteine. Lysates wereimmunoprecipitated with antibodies to p56^(lck) and 4-1BB and labeledwith [γ-³²P] ATP by the in vitro kinase reaction. The positions ofp56^(lck) and 4-1BB are indicated to the right. Total HeLa celll lysateswere immunoblotted with antibody top56^(lck) assay thep56^(lck)expression. FIGS. 32b and 32 c show an immune complex kinase assay.Aliquots of the saline lysates were immunoprecipitated withantip56^(lck) serum (FIG. 32b) or 53A2 (FIG. 32c) followed by the invitro kinase reaction with [γ-³²P] ATP as described in the legend toFIG. 30. 1×106 HeLa celis were grown to 80% confluency on 100-mm petridishes in DMEM containing 10% FBS and antibiotics. Cells were infectedwith 1×10⁸ pfu of vaccinia virus expressing T7 RNA polymerase (136) 30min before transfection. DNA was transfected in liposomes (Lipofectin,GIBCO, BRL). 15 μg DNA and 50 μl liposome diluted into 5 ml serum-freeDMEM were incubated in 12×75 mm polystyrene tubes at room temperaturefor 15 min.

[0259] The expression of p56^(lck) was assayed by immunoblotting of atotal cellular lysate with anti-p56^(lck) (FIG. 32). The associationbetween 4-1BB and p56^(lck) was examined by the in vitro kinase-labelingof the 53A2 immune complex as in the baculoviral expression system (FIG.32). 4-1BB, wild-type p56^(lck) oy the combination of 4-1BB andp56^(lck) of either the wild-type or mutant phenotypes, C1 or C2, wereexpressed in HeLa cells (FIG. 32c). 53A2 coimmunoprecipitated p56^(lck)with 4-1BB only when 4-1BB was coexpressed with the wild type p56^(lck).The coimmunoprecipitation of p56^(lck) with 4-1BB by 53A2 was notobserved when 4-1BB was coexpressed with either of the C1 and C2p56^(lck) proteins (FIG. 32c). As shown in previous studies in thebaculoviral expression system, the fact that the presence of 4-1BB wasrequired for 53A2 to coimmunoprecipitated p56^(lck) ruled out thepossibility of cross-reactivity of the 53A2 mAb with p56^(lck) 4-1BBassociated specifically with the wild-type p56^(lck) and mutation of cys20 or cys 23 of p56^(lck), crucial for binding to CD4 or CD8, prohibitedinteraction with 4-1BB in HeLa cells. The disruption ofcoimmunoprecipitation of mutated p56^(lck) was not due to lack ofexpression of el˜her p56^(lck)- or 4-1BB as proven by comparable levelsof expression of immunoblotted p56k (FIG. 32a) and immunoprecipitated[3SS] 4-1BB (FIG. 32c). FACS staining with 53A2 of HeLa cellstransfected with 4-1BB and p56^(lck) wild-type, C1 or C2 Ir˜dicated thatthe expression levels of cell surface 4-1BB were comparable (data notshown). Since the detection of p56^(lck) was dependent on its in vitrolabeling efficiency, the specific autophosphorylation activity of thewild-type and mutant p56^(lck) proteins was compared. Cell lysatescontaining each p56^(lck) recombinant protein were immunoprecipitatedwith anti-p56^(lck) serum and autophosphorylated. As shown in FIG. 32b,the band intensities of wildtype and mutant p56^(lck) were comparable.Thus, all the p56^(lck)-mutants tested showed similarautophosphorylation activities and were expressed at similar levels inthe cells. This suggests that the specific kinase activity of mutantp56^(lck) was almost identical to that of wild-type p56^(lck).Therefore, it was concluded that the failure of the mutant p56^(lck) C1or C2, to associate with 4-1BB was due to the absence of criticalcysteine residues but not due to a defect in kinase activity caused bymutation.

[0260] The fact that 4-1BB physically associates with p56^(lck) and thatits interaction requires the same two cysteines in p56^(lck) alsocritical to binding to CD4- or CD8 suggests that 4-1BB may compete withCD4 or CD8 for coupling to p56^(lck)-Expression of 4-1BB is highlyinducible during T-cell activation, in contrast to that of CD4 or CD8,which is constitutively expressed. Therefore, it is conceivable that theexact intracellular localization of p56^(lck) in T cells may be modifiedduring cell activation with increasing amounts of competing bindingsites for p56^(lck) provided by 4-1BB. At present, the relative bindingaffinity of p56^(lck) to 4-1BB or to CD4 or CD8 in unknown as well asthe relative amount of p56^(lck) binding with 4-1BB or CD8 in CTLL-2cells.

[0261] The CD4- or CD8-p56^(lck) association has been shown essentialfor the optimal response of T cells to antigen (124). p56^(lck)incorporated in the antigen-receptor complex through the juxtapositionof CD4 or CD8 coreceptors might serve to relay and modify TCR signals tothe cell interior (125). This signaling relay may be attributed to thephysical proximity of CD4- to TCR, caused by either crosslinkingreagents or antigen presentation by MHC Class II molecules (126). Atthis time, it is still possible that 4-1BB may assemble with the CD4- orCD8-p56^(lck) complex rather than act as a competitor of CD4- or CD8.The association of p56^(lck) with 4-1BB may expose this kinase to otherregulatory proteins and substrates. Likewise, the regulation ofp56^(lck) by CD4 or CD8 might be modified once 4-1BB is expressed.Recently, the CD4 or CD8-p56^(lck) complex has been found to includeadditional proteins (127) such as a GTP-binding protein, p32 (128) orRaf-I-related protein, p110 (129), as an assembled complex. Theassociation ofp56^(lck) and CD4 may be regulated by intracellularsignals, for the protein kinase C-activator, TPA, caused theinternalization of CD4 (130) as well as the dissociation of CD4- fromp56^(lck) (131) If the 4-1BB-p56^(lck) association is regulated byT-cell immunoregulatory factor, the outcome would provide a furthermechanism for regulating T-cell growth and subsequent immune function.

[0262] The 53A2 monoclonal antibody enhanced the proliferative responseof anti-CD3-stimulated murine splenic T cells. A recent study (132)suggested that 4-1BB contains N-linked and O-linked carbohydyrates aswell as sialic acid, and interacts with various extracellular matrixcomponents. The possibility that stimulatory signaling through 4-1BBinvolves an alteration in p56^(lck) mediated protein tyrosinephosphorylation is being pursued. Since not all cellular p56^(lck) isassociated with CD4- or CD8, and since p56^(lck) is involved inthymocyte development (77, 133), it is plausible that the4-1BB-p56^(lck) complex may be involved in regulating early and/or lateantigen receptor induced-signals, or possibly intrathymicdifferentiation of T cells.

[0263] 4-1BB T-Cell Antigen Binds to Mature B Cells and Macrophages, andCostimulates Anti-μ Primer Splenic B Cells

[0264] 4-1BB is expressed on activated murine T cells and may functionas an accessory signaling molecule during T-cell activation. In order toidentify putative cell-surface 4-1BB ligands, a fusion proteinconsisting of the extracellular domain of 4-1BB fused to human placentalalkaline phosphatase (4-1BB-AP) was construed. Alkaline phosphataseactivity was used as an indicator of the relative amount of bound 4-1BB.The 4-1BB-AP fusion protein was used in a semi-quantitative bindingassay and for in situ staining to determine if a membrane-bound ligandexists for 4-1BB. These studies indicate that 4-1BB-AP bound to thesurface of various mature B- and macrophage-cell lines. The binding wassaturable, and inhibited by recombinant, soluble 4-1BB protein. 4-1BB-APbound at low levels or not at all to T-cell lines (non-activated andanti-CD3-activated), pre-B-cell lines, an inunature macrophage cellline, a glial tumor-cell line, HeLa cells, or COS cells. ln addition,4-1BB-AP bound to F(ab′) anti-μ-activated primary culture B cells butnot to anti-CD3-activated primary culture T cells. Although signalsdelivered through 4-1BB may influence T-cell activation, the resultspresented here, suggest that 4-1BB may, in addition, function as aregulator of B-cell growth. The addition of paraformaldehyde-fixed SF21cells expressing recombinant 4-1BB, synergized with F(ab′), anti-μ ininducing splenic B-cell proliferation.

[0265] Although the interaction of the T-cell receptor complex with itsspecific peptide/MHC on antigen presenting cells (APCs) is required foreffective collaboration between these cells, it is becoming clear that aplethora of other cell-cell interactions is also necessary. Thesereceptor-counter receptor interactions are being studied extensively inmodel systems of thymus-dependent B-cell activation; it has beenproposed that B-cell/T-cell interactions result in a co-directionalrelay of biochemical signals which are responsible for cell cyclecommitment (78). Receptor-ligand pairs between B and T cells presentlycharacterized are the LFA-1/ICAM-1 (79) and CD2/LFA-3 (80) receptorpairs involved in cell adhesion; the Lyb2/Lyl (81) the B7/CD28 (82) orthe B7/CTLA4 (83) involved in T-cell costimulation; and the CD4O/CD4OL(84,85)which is involved in B-cell proliferation and differentiation.

[0266] 4-1BB is a member of the Nerve Growth Factor Receptor (NGFR)superfamily (89,92) and is expressed on the surface of activated CD4⁺and CD8₊ murine T cells. The NGFR superfamily members are characterizedby the presence of three to six patterns of a cysteine-rich motif, eachconsisting of about 40 amino acids in the extracellular region of themolecule. These cysteine-rich motifs may result in receptors possessingextracellular domains with similar tertiary structures and thus similarligand-binding properties. In contrast, the cytoplasmic tails of thesereceptors are unique, predicting that different biochemical signalscould be delivered intracellularly once the ligand binds.

[0267] The majority of cell-surface 4-1BB exists as a 55 kDa. dimer onthe surface of T cells. 4-1BB may act as an accessory signaling moleculeduring T-cell activation; the addition of an anti-4-1BB-monoclonalantibody, 53A2, resulted in an enhancement of the anti-CD3-inducedproliferation of purified splenic T cells. Although the actualbiochemical signals delivered through 4-1BB are not known, it has beenshown that 4-1BB is coupled to the tyrosine kinase, p56^(lck),suggesting that this kinase may play a role in transmitting signalsdelivered through 4-1BB (under publication).

[0268] At present, in the NGFR superfamily, only ligands for the NGFR(105), Tumor necrosis factor receptors 1 and 11 (48), and CD4O 84,85)have been identified. To identify 4-1BB cell-surface ligands, a 4-1BBfusion protein, 4-1BB-AP, which consists of the extracellular domain of4-1BB fused to human placental alkaline phosphatase was generated. The4-1BB-AP fusion protein bound to various mature B-and macrophage-celllines. In addition, data presented in this report suggest 4-1BB may actas a signaling molecule for anti-μ-primed B cells. The identification ofcell surface and/or soluble 4-1BB ligands will be essential inunderstanding the physiological role of 4-1BB during T-cell-APCinteractions.

[0269] Materials and Methods.

[0270] Mice. Female Balb/c mice were obtained from Harlan Indianapolis,Ind.) and used at 6-10 weeks of age. Cells. NIH-3T3 cells weremaintained in DMEM containing 7% fetal bovine serum (FBS) (DMEM-CM).2PK-3, WEHI-231, and 70Z/3 cells were maintained in DMEM containing 10%FBS, 50 μM 2-mercaptoethanol (2-ME) and antibiotics. HeLa and COS cellswere maintained in RPMI-1640 containing 10% FBS, and antibiotics(RPMI-CM). A20, 230, WEHI-3, and P388D, cells were maintained in RPMI-CMsupplemented with 1 mM sodium pyruvate, 1 mM nonessential amino acids,and 2-ME (RPMICM⁺). The BCL₁-5B₁b (BCL₁) B-cell leukemic cells weremaintained in RPMI-CM⁺ with 15% FBS. The PU5-1.8 and RAW-264.7 cellswere maintained in RPMI-CM containing 2-ME. The WR19M.1 cells weremaintained in RPMI-CM+ containing 10% horse serum and 5% FBS. CTLL-2(CD8⁺), CTLL-R8 (CD8⁺), D 10.G4 (CD4⁺) (106), and F 1 (CD4⁺) cells, weremaintained in RPMI-CM containing 10% Rat Con A supernatant (106).Spodoptera frugiperda (SF-21), an insect cell line was grown insynthetic serum-free Ex-cell 400 medium (JRH Biosciences) containingantibiotics at 27° C. These cells were infected with wild-typebaculoviruses or baculoviruses expressing either the recombinantfull-length 4-1BB or the recombinant extracellular domain of 4-1BB(rs4-1BB), as previously described (53). (The 230 and WR19M. 1 cellswere a generous gift of Dr. Pat Stuart, and the D10.G4 and F1 T-cellclones were a generous gift from Dr. Scott Bryson, both of theUniversity of Kentucky, Lexington, Ky. The PU5-1.8, P388D₁, and C₆ cellswere a generous gift of Dr. Randy Rosenthal, University of Indiana,Indianapolis, Ind. The BCL₁-5B₁b cells were the kind gift of Dr. RomanDziarski, Northwest Campus, Indiana University, Gary Ind. The otherlymphoma cell lines were obtained from American Type CultureCollection.)

[0271] Production of the 4-1BB-AP fusion protein. The 5′ portion of the4-1BB cDNA including sequences encoding the original signal peptide andthe entire extracellular domain, was amplified by the polymerase chainreaction (PCR) (99). For correctly oriented cloning, a Hind III site onthe 5′ end of the forward primer and a Bgl II site on the 5′end of thereverse primer were created. The Hind III-Bgl II 4-1BB fragment wasinserted into the mammalian expression vector AP-tag-1, upstream of thecoding sequence for human placental alkaline phosphatase (Ap) (113).(The AP-tag-1 vector was a kind gift of Dr. John Flanagan, HarvardUniversity, Cambridge, Mass.). Sequence analysis of the fusion regionconfirmed that the 4-1BB and AP sequences were joined in frame. The4-1BB-AP plasmid, linearized with Cla I, was cotransfected with thelinearized-selectable marker plasmid, pSV7neo, by the calcium phosphatecoprecipitation method. After selection in 500 μg/ml G418, resistantcolonies were picked and expanded. Clones were subsequently screened forsecretion by assaying for AP activity. Supernatant from one clone,4-1BB-AP-2, which produced the 4-1BB-AP fusion protein, showed highlevels of alkaline phosphatase activity; 738 OD units/hr/ml. Whendetermining total 4-1BB-AP or AP activity, serial dilutions wereperformed so that AP activity was measured at non-saturating levels. The4-1BB-AP or AP was then diluted accordingly so equivalent levels of4-1BB-AP or AP activity were added to each sample. DMEM-CM containingpurified human placental AP (Sigma, St. Louis Mo.) was utilized as abackground control in all experiments.

[0272] Western Analysis of 4-1BB-AP. Protein samples were resolved byelectrophoresis on a 12% SDS-polyacrylamide gel, and transferredelectrophoretically onto an Immobilon-P membrane (Millipore, Bedford,Mass.). The membranes were blocked to prevent nonspecific antibodybinding by incubating in 5% nonfat dry milk in TBST (50 mM Tris HCI, pH7.4, 0.15 M NaCl and 0.05% Tween-20) for 1 hr at room temperature. Themembrane was then incubated with an anti-4-1BB peptide serum(anti-4-1BB-0) at room temperature for 1 hour with gentle agitation. The4-1BB-0 oligopeptide was a 11-mer from amino acids 105-115 of thededuced 4-1BB sequence (89). After four washes with TBST, the membraneswere incubated with a secondary antibody against rabbitIgG(H+L)-alkaline phosphatase conjugate at 1:1000 dilution. The reactivebands were visualized by incubating the membrane with chromogenicsubstrates, p-nitroblue-tetrazolinum chloride (NBT) and5-bromo-4-chromo-3-indolyl-phosphate (BCIP) in 0.1 M Tris, pH 9.5, 0.1 MNaCl, and 5 mM MgCl².

[0273] 4-1BB-AP Ouantitative Binding Assay. The method of Flanagan andLeder (113) was used with some modification to assay for cell-bound4-1BB-AP. Cells were washed with HBHA buffer [Hank's balanced saltsolution with 0.5 mg/ml BSA, 0.1% NaN³, 20 mM HEPES (pH 7.0)]. Two×10⁶cells per sample were incubated in eppendorf tubes with DMEM-CMcontaining equivalent levels of 4-1BB-AP or AP activity. Allsupernatants contained 0.1% NaN³. Samples were incubated for 60-90minutes at 4° C. with slow rotation. The cells were pelleted and washedthree times with HBHA buffer. The cells were then lysed in 375 μl 1%Triton X-100, 10 mM Tris-HCl (pH 8.0). The lysate was vortexedvigorously and the nuclei removed by centrifugation at 10,000×g for 5minutes. This assay takes advantage of the fact that this particular APisozyme is stable at 65° C. whereas endogenous phosphatases areinactivated at this elevated temperature. Supernatants were placed inclean tubes and heated for 0.10-15 minutes at 65° C. to inactivatebackground cellular phosphatases.

[0274] Alkaline Phosphatase assay. Each sample was assayed for APactivity in triplicate. Alkaline phosphatase activity was measured byincubating 100 μl of heat-inactivated supernatants with 100 μl of 2X APbuffer (1X=1M diethanolamine pH 9.8, 0.5 mM MgCl², 10 mM homoarginine,0.5 mg/ml BSA and 12 mM P-nitrophenyl phosphate), and measuring theOD₄₀₅. (114).

[0275] In situ analysis of 4-1BB-AP binding. Binding and washing weredone as described above for the 4-1BB-AP quantitative binding assay. Thecells were then spun onto slides by a cytospinner (Cytospin 2, Shandon,UK) and subsequently fixed for 30 seconds in 60% acetone, 3%formaldehyde, 20 mM HEPES (pH 7.5), washed twice for 5 minutes each in150 mM NaCl, 20 mM HEPES (pH 7.5). The slides were then floated on 65°C. water bath for 10 minutes to inactivate cellular phosphatases. Afterrinsing with 100 mM Tri-HCl (pH 9.5), 100 mM NaCl, 5 mM MgCl², the cellswere stained for 72 hours in the same buffer containing 10 mML-homoarginine, 0.17 mg/ml BCIP, 0.33 mg/ml NBT for color development.Methyl green was used as a counter stain for nuclei. The slides weremounted using glyceroi-polyvinyl alcohol aqueous mounting medium.

[0276] Purification of the 4-1BB-AP fusion protein. Culture supernatantsfrom NIH-3T3 transfectants (DMEM-CM) containing 4-1BB-AP fusion Proteinwere initially fractionated and concentrated by ammonium sulfateprecipitation (60% saturation). Precipitates were resuspended at 10% ofthe original volume in 40 mM Tris, pH 8.0 and dialyzed overnight againstthe same buffer. The proteins were fractionated by ion exchangechromatography on Q-Sepharose fast flow (Pharmacia, Piscataway, N.J.) atpH 8.0, Fractions containing 4-1BB-AP were identified by AP activity andan immunoblot assay as described above. 4-1BB-AP containing fractionswere pooled and dialyzed overnight against 40 mM Tris 150 mM NaCl, pH7.5. The dialyzed proteins were affinity purified using an anti-humanplacental alkaline Phosphatase row affinity anti-AP antibody, MedixBiotech, Foster City, Calif.)-affinity column (Affi-gel HZ, BioRad,Richmond, Calif.) as described by Flanagan and Leder (113), with theexception that the elution buffer was adjusted to pH 4.5. Fractionscontaining 4-1BB-AP fusion protein were identified by AP and immunoblotassays and their purity was assessed by Coomassie blue staining of a 10%SDS-polyacrylamide gel.

[0277] Costimulation of splenic B cells with paraformaldehyde-fixed SF21cells expression recombinant 4-1BB. Spleens were removed asepticallyfrom mice sacrificed by cervical dislocation, and teased into a singlecell suspension in 5% FBS-RPMI-1640. Small resting B cells were purifiedby treatment with anti-L3T4 (clone GKI.5), anti-Thy 1.2 (clone 13-4.6)followed by baby rabbit complement (PelFreeze, Brown Deer, Wis.). The Bcells subsequently were fractionated by centrifugation at 2000×g for 30min at 4° C. on a four step gradient consisting of 80% (1.08 g/ml), 70%(1.07 g/ml), 60% (1.06 g/ml), and 50% (1.05 g/ml) Percolt solutions.Resting B cells were recovered from the interface between the 60% and70% Percoll layers. These B cell preparations were 90% L3T⁻and wereunresponsive to Con A. SF21 cells were infected for 36 hours withwild-type baculoviruses (SF2 1-wt) or recombinant baculovirusesexpressing the full length 4-1BB (SF21-4-1BB). After infection, cellswere washed twice in HBSS, fixed with 1% paraformaldehyde for 15 minutesat room temperature, and washed once with HBSS. The fixed cells werethen resuspended in 25 ml of 0.1 M lysine for 30 minutes at roomtemperature, and washed once in RPMI-CM. Five×104 fixed-SF21 cells(SF21-wt or SF21-4-1BB) were incubated with 1×10⁵ B cells per well in 96well microtiter plates. After 72 hours of stimulation, cultures werepulsed with 1 μCi per well ³H-thymidine for 16 hours. Cultures wereharvested onto glass fibre filters, and ³H-thymidine incorporation wasmeasured in a liquid scintillation counter.

[0278] Results.

[0279] Expression of the 4-1BB-Alkaline Phosphatase (4-1BB-AP) fusionprotein. In the mammalian expression vector, APtag-1, the 5′extracellular domain of a receptor can be fused with AP (113). Thisintrinsic AP activity can be used to monitor the binding of a solublereceptor to the cell surface with high sensitivity in bothsemi-quantitative and in slit assays. The 5′ portion of the 4-1BB geneconsisting of sequences encoding the signal peptide and the entireextracellular domain immediately before the first hydrophobic amino acidof the transmembrane region (4-1BBs), was inserted upstream of theentire coding sequence of AP in the APtag-1 vector. The 4-1BB-APexpressing plasmid, 4-1BB-AP-TAG, and pSV7neo were cotransfected intoNIH-3T3 cells and G418-resistant clones were selected. Northern andWestern analyses, and the AP assay were used to select for clones thatproduce high levels of 4-1BB-AP in the supernatant. Ananti-4-1BB-immunoreactive band of approximately 85 kDa. FIG. 33 showsthe expression of the 4-1BB-AP fusion protein and rs4-1BB. Supernatantsfrom rs4-1BB baculovirus-infected SF21 cells lane 1), Neor-N13T3-cells(lane 2), and 4-1BB-AP/Neor-NIH-3T3 cells lane 4) were analyzed by aWestern blot which was probed with the anti-4-1BB-0 serum. Lane 3represents Coomassie blue-pre-shined molecular weight standards. Lane 5represents 10 μg purified human placental alkaline phosphatase stainedwith Coomassie blue. The 4-1BB-AP fusion protein is 85 kDa. lane 4), andthe AP protein is 67 kDa. (lane 5) was secreted into the supernatant.The 85 kDa. protein was recognized by an anti-4-1BB peptide serum(4-1BB-0) (FIG. 33) and by an anti-4-1BB monoclonal antibody, 53A2 (datanot shown). The purified 85 kDa. fusion protein possessed AP activity,and consisted of the 67 kDa. AP protein (FIG. 33, lane 5) and the 1 8-23kDa. rs4-1BB protein (FIG. 33, lane 1).

[0280] Quantitative Analysis of 4-1BB-AP binding to lymphoid andnonlymohoid cell lines. Measurement of AP activity as an indicator ofbound 4-1BB-AP provided a reliable method for an initial determinationof the relative amount of 4-1BB-AP bound to one cell as compared toanother cell type. Two×16 cells were incubated in 1 ml of DMEM-CMcontaining equivalent levels of 4-1BB-AP or AP activity (700 ODunits/hr/ml). In all experiments, cell viability was ≧95% as determinedby trypan blue exclusion. The cells were washed, lysed, and assayed forAP activity. The relative levels of 4-1BB-AP bound were determined bysubtracting background levels of AP bound from levels of 4-1BB-AP boundto each cell type. The range of AP bound routinely ranged from an OD⁴⁰⁵,0.1 to 0.45.

[0281]FIGS. 34a-c show the quantitative analysis of 4-1BB-AP binding tolymphoid- and nonlymphoid-cell lines. In Experiments I and II 2×10⁶cells were used per sample. In Experiment III, 2×10⁶ 2PK-3 cells, and6×10⁶ purified splenic B cells or T cells, or FI-T cells were used persample. Samples were incubated with 4-1BB-AP or AP, washed, and assayedfor AP activity.

[0282] The highest level of 4-1BB-AP binding was observed with the IgG⁺B-cell A20 and 2PK-3 lymphomas (FIG. 34a). LPS (15 μg/ml) or acytokine-rich supernatant (1:10) from D1O.G4 cells (D1Os) increased thelevel of 4-1BB-AP binding to A20 cells. Increased levels of 4-1BB-APbinding were also observed when 2PK-3 cells were stimulated with thesereagents (data not shown). In contrast, WEHI-3, a myelomonocytic cellline, or 230, a pre-B cell line, bound low levels of 4-1BB-AP, and whenstimulated with LPS for 24 hours did not show increased binding to4-1BB-AP (FIG. 34b) and data not shown). TPA caused a slight downregulation of 4-1BB-AP binding sites on A20 cells. The CD4⁺ T-cellclones, F1 and DIO.G4, and the CD8⁺ T-cell line, CTLL-2, boundnegligible levels of 4-1BB-AP (FIG. 34a). Activation of CTLL-2 cells byanti-CD3 did not increase binding of 4-1BB-AP to any significant degree.In data not shown, 4-1BB-AP also did not bind to CTLL-R8 cells or toanti-CD3-activated F1 or D10.G4 cells. 4-1BB-AP did not bind tononlymphoid cells such as C⁶ (rat glial tumor), HeLa (human epitheliodcarcinoma), or COS cells (monkey SV-40-transformed fibroblast) (FIG.34a).

[0283] Most notably, the IgG⁺ B cells (2PK-3, A20) bound higher levelsof 4-1BB-AP than the IgM⁺ B cells (WEHI-231, BCL¹) or the IgG-pre-Bcells (230) (FIG. 34b), In other experiments, another IgG-pre-B cellline, 70Z/3, bound levels of 4-1BB-AP comparable to 230 pre-B cells(data not shown). While the macrophage cell line, WR19M.1, bound4-1BB-AP at levels similar to those bound by IgM⁺ B-cell lines, themacrophage cell lines, PU5-1.8 and RAW 264.7 bound 4-1BB-AP at levelssimilar to those of the IgG⁺ B-cell lines (FIG. 34b). Not all lymphoidcell lines bound to 4-1BB-AP, for P388D, cells (FIG. 34a) and WEHI-3cells, (FIG. 34b), bound neglible levels of 4-1BB-AP. These data suggestthat a 4-1BB-binding site potentially exists on the surface of matureAPCs such as macrophages and B cells; it is possible 4-1BB may regulatesome aspect of APC function.

[0284] It was also determined if 4-1BB-AP bound to normal B cells.Resting splenic B cells were purified and found to bind low levels of4-1BB-AP (FIG. 34c). B cells stimulated with Goat anti-mouse F(ab′),anti-μ (anti-μ) (Cappel, Durham, N.C.) for 24 hours, also exhibited lowlevels of 4-1BB-AP binding, while at 60 hours post-stimulation withanti-μ, binding increased 4 fold compared to nonstimulated B cells. Lowlevels of 4-1BB-AP bound to resting T cells purified as previouslydescribed above by a nylon wool column; this binding may be attributedto small levels of activated B cells or macrophages in the culture.However, no increased binding was observed when 4-1BB-AP was added to Tcells stimulated with anti-CD3 for 24 and 60 hours. This studydemonstrated that selective binding of 4-1BB-AP to B-cell lines comparedto T-cell lines was not merely a characteristic of transformed B-celllines, since activated primary culture B cells also possessedsignificantly higher levels of 4-1BB-AP binding than activated primaryculture T cells.

[0285] In data not shown, B-cell lymphomas were washed in an acid buffer(10 mM sodium citrate, 140 mM NaCI, 0.1% bovine serum albumin, pH 4.0)prior to the addition of 4-1BB-AP to remove bound ligands from the cellsurface (115). 4-1BB-AP bound to cells washed with HBHA buffer or acidbuffer at identical levels (data not shown). This ruled out thepossibility that 4-1BB-AP was binding to proteins extrinsically attachedto the cell surface.

[0286] Characterization of 4-1BB-AP binding to the A20 B-cell Lymphoma.A20 or D10.G4 cells were incubated with increasing amounts of 4-1BB-APto determine the concentration required to saturate 4-1BB-AP bindingsites. Saturation was reached at approximately 375 ng/ml (4.4 nM)4-1BB-AP (FIG. 35a). AP activity was indicative of the saturability of4-1BB-AP binding sites since the AP substrate, pnitrophenyl phosphate,which was present in excess.

[0287]FIGS. 35a and 35 b show the characterization of 4-1BB-AP bindingto A20 B-cell lymphoma cells. FIG. 35a shows the saturation of 4-1BB-APbinding sites on A2O cells. Two×10⁶ A2O or D1O cells were incubated withincreasing concentrations of 4-1BB-AP, washed, and assayed for APactivity. FIG. 35b shows the inhibition of 4-1BB-AP binding to A2O cellsin the presence of rs4-1BB. Two×10⁶ A2O cells were preincubated with orwithout 20 μg/ml rs4-1BB for 30 minutes on ice. Samples were thenincubated with 4-1BB-AP or AP. The cells were washed and assayed for APactivity.

[0288] The specificity of 4-1BB-AP binding was analyzed bypre-incubating A20 cells with rs4-1BB prior to the addition of 4-1BB-AP.For expression of rs4-1BB, SF21 cells were infected with recombinantbaculoviruses containing the sequence encoding the extracellular regionof 4-1BB; rs4-1BB was purified from supernatants of infected SF2 1 cellsas described above. A20 cells were preincubated in 1 ml of PBS, 2% BSA,0.1% sodium azide with or without 20 μg/ml (50 fold excess) rs4-1BB for30 minutes on ice. 4-1BB-AP was subsequently added b rs4-1BB-treated andnontreated A20 cells at saturating levels (FIGS. 35 −375 ng/ml). WhenA20 cells were preincubated with 20 μg/ml (rs4-1BB, the binding of4-1BB-AP to A20 cells was blocked by approximately 70% (FIG. 35).

[0289] A Western analysis of rs4-1BB (FIG. 33) indicated that rs4-1BBexisted as several different immunoreactive species; this may be due todifferential posttranslational modifications indicative of thebaculovirus system. For this reason, the binding affinity of rs4-1BBcompared to 4-1BB-AP may differ. These data, in conjunction withexperiments demonstrating selective binding of 4-1BB-AP to mature Bcells and macrophages (FIG. 34), suggest 4-1BB-AP binds specifically.

[0290] In Situ Staining of 4-1BB-AP Protein Bound to A20 cells. Thebinding of 4-1BB-AP to A20 cells was next examined by in situ stainingin order to assess the distribution of bound 4-1BB-AP. A20 or DIO.G4cells were incubated with equal levels of 4-1BB-AP or AP activity. Afterwashing, the cells were spun onto slides with a cytospinner, fixed, andanalyzed for AP activity. A20 cells incubated with 4-1BB-AP showedcell-surface staining, representative of AP activity. A20 cellsincubated with AP alone did not exhibit any observable staining. On thecontrary, D1O,G4 cells incubated with 4-1BB-AP or AP alone showed noobservable cell-surface staining (data not shown). In all cases, thenuclei were counter-stained with methyl green. These data indicate A20cells express binding sites for 4-1BB-AP on the cell surface.

[0291] Costimulation of primed splenic B cells withparaformaldehyde-fixed sf21-4-1BB cells. Since 4-1BB bound toanti-μ-treated B cells, experiments were performed to determine whether4-1BB may deliver growth-inducing signals to B cells. Small resting Bcells were stimulated with various concentrations of anti-μ in theabsence or presence of paraformalehyde-fixed SF21-4-1BB or SF21-wtcells.

[0292]FIG. 36 shows the costimulation of anti-r-primed B cells withfixed-SF21-4-1BB cells. 1×10⁵ splenic B cells were incubated in mediumalone (C) or stimulated with 0.1-IO μg/ml of anti-μ in the absence orpresence of 5×10⁴-SF21-wt or SF21-4-1BB cells. At 72 hours, cultureswere pulsed with 1 μCi per well ³H-thymidine or 16-18 hours, harvested,and ³H-thymidine was measured by liquid scintillation counting.

[0293] FACS analysis indicated that at 2 days postinfection, >80% of theinfected SF21 cells expressed 4-1BB on the cell surface (data notshown). 4-1BB functioned as a costimulator of B-cell proliferation whencells were incubated with optimal (10 μg/ml) or suboptimal (0.1-1.0μg/ml) concentrations of anti-i.

[0294] SF21 cells did not exhibit efficient costimulator activity wheninfected with wild type baculoviruses (FIG. 36, compare SF21-wt toSF21-4-1BB stimulations). SF21-4-1BB did not synergize with TPA,ionomycin, TPA plus ionomycin or suboptimal concentrations of LPS (datanot shown) in inducing B-cell proliferation. A synergistic response alsoresulted when B cells were stimulated with SF2i+1BB and D1Os (data notshown). This study demonstrates that 4-1BB is capable of delivering asignal(s) to B cells which synergizes with those generated by theaddition of anti-μ.

[0295] Discussion

[0296] The characterization of receptor.counter receptor interactionsduring contact between T cells and B cells or other APCs, is only in itsinfancy. 4-1BB may represent another cell-surface molecule involved in Tcell-APC interactions. The 4-1BB-AP fusion protein specifically bound tomature B-cell lines, anti-μ-activated primary B cells, and maturemacrophage-cell lines. 4-1BB-AP bound at low or insignificant levels toimmature B- and macrophage-cell lines, T-cell clones, T-cell lines,primary culture T cells, and various nonlymphoid-cell lines. Since4-1BB-AP binds to mature B cells and macrophages, it is possible thatsignals delivered upon 4-1BB binding may modulate APC functions in someway. This possibility remains to be explored.

[0297] Chalupny and colleagues (132) have proposed that 4-1BB Rg, afusion protein consisting of the extracellular domain of 4-1BB and theFc region of human IgG₁, bound to the extracellular matrix (ECM). Thehighest levels of 4-1BB Rg binding was to human vitronectin. In data notshown, an ELISA was performed using 4-1BB-AP and human vitronectin(Yelios Pharmaceuticals/GIBCO-BRL, Grand Island, N.Y.) immobilized at0.007 μg-10 μg per well on microtiter plates. No binding of 4-1BB-APbased on AP activity was observed. To rule out the possibility that4-1BB-AP was binding to proteins extrinsically attached to the cellsurface (possible extracellular matrix components), B-cell lymphomaswere washed in acid conditions prior to the binding assay (115).4-1BB-AP still bound specifically to mature B-cell lymphomas (data notshown). We are currently determining whether a 4-1BB-ligand specificallyexpressed on B cells and macrophages exists, and whether 4-1BB-AP maybind to the ECM under particular binding conditions. It is possible thatthe ECM could facilitate the binding of 4-1BB to a specific cell-surfaceligand.

[0298] B cells and helper T cells interact with each other throughreceptors on B cells binding to their specific counter-receptors on Tcells. It is thought that this interaction results in a cascade ofbiochemical signaling relays between these two cell types (78). As thisinteraction proceeds, these cells become committed to enter the S phaseof the cell cycle. Initial interactions between TCR and CD4 on T cells,and processed antigen-MHC II on B cells, do not result in B cellscapable of entering the cell cycle (116). However, studies from in vitrosystems suggest that once Th cells are stimulated, they express newlysynthesized or modified cell-surface molecules capable of inducing Bcells to enter the cell cycle (22, 23). This T-cell function is notantigen-specific or MHC-restricted (24). In addition, soluble factorsare not required for the activated Th induction of B-cell activation(21). Once B cells enter the cell cycle, IL-4 induces B cells toprogress from G₁ to S phase. The ability of activated T cells or T-cellmembranes to promote the entry of B cells into the cell cycle can beblocked by either cycloheximide or cyclosporin A treatment (117, 118).These newly expressed membrane proteins appear to be “lymphokine-like”in their induction characteristics.

[0299] 4-1BB has expression properties which meet the requirements of aB-cell costimulator. 4-1BB is inducible by anti-CD3 or TCR-mediatedT-cell stimulation, and its expression is sensitive to cyclosporin A aswell as cycloheximide treatment (90). Interestingly,paraformaldehyde-fixed SF21-4-1BB cells, synergized with anti-μ ininducing B-cell proliferation. The costimulation of splenic B cells bySF21-4-1BB occurred at optimal (10 μg/ml) and suboptimal (1.0-0.1 μg/ml)doses of anti-μ. The addition of SF21-4-1BB cells to resting B cells,did not result in significant B-cell proliferation. SF21-4-1BB cells didnot synergize with TPA or ionomycin, or suboptimal concentrations of LPSin inducing B-cell proliferation (data not shown).

[0300] Although the baculovirus system has been used to express largeamounts of recombinant soluble proteins, this system may be utilized forthe expression of recombinant cell-surface proteins. The baculovirusinfection provides a convenient means to express uniformity high levelsof recombinant protein on a per cell basis. It is noteworthy, that theaddition of SF2 1 cells alone did not result in significant levels ofcostimulation. This can be a potential problem when using cos- or L-celllines which can exhibit strong costimulator activity on their own.

[0301] Another member of the NGFR superfamily, CD4O, is expressed on Bcells and interacts with gp39, a molecule expressed on activated Tcells. The cDNAs encoding the murine (84) and human (26) gp39 proteinshave been cloned; this cell surface molecule is a type II membraneprotein with homology to tumor necrosis factor. Noelle et al. (85) foundthat a CD4O-immunoglobulin fusion protein, is capable of blocking Tcell-induced B-cell proliferation and differentiation in adose-dependent manner. Armitage et al (84) have isolated, a cDNA formurine gp39 and showed that gp39 could induce B-cell proliferation inthe absence of co-stimuli, and result in IgE production in the presenceof IL-4-. Hollenbaugh et al. (120) have shown that COS cells transfectedwith human gp 39 can synergize with either TPA or anti-CD2O in inducinghuman B-cell proliferation and is able to stimulate B cells without acostimulator only at low levels. These data indicate that CD4O may beone of the B-cell-surface molecules that transmit signals duringphysical contact with T cells.

[0302] Cell-surface receptors communicate with their external milieu byinteracting either with soluble factors or other cell surface moleculesexpressed on neighboring cells. The role of biochemical signalsdelivered by cell-cell contact versus those delivered by soluble factorsinteracting with cell surface receptors is not clear. The NGFRsuperfamily is unusual for the TNFR I and II as well as the NGFR bind tomore than one ligand. The TNFRs I and II both bind to TNF-α and TNF-R(48). The NGFR binds to NGF, brain-derived neurotrophic factor, andneurotrophin-3 (105).

[0303] In addition, one ligand may function as both a cell surface andsoluble ligand. Recent evidence on the CD4-0 ligand, gp39, suggests thatthis ligand can exist as a membrane bound as well as a soluble ligand(121). It may be possible that 4-1BB is secreted and interacts with Bcells in a soluble form as well as a membrane bound form. A member ofthe NGFR receptor family, CD27, which is expressed on T cells, issecreted in addition to being expressed on the cell surface (122). It isalso possible that more than one 1 ligand (soluble and cell surface) maybind to 4-1BB.

[0304] In the present study, we have shown that 4-1BB-AP binds to thesurface of a variety of B-cell and macrophage-cell lines, and activatedprimary culture B cells. 4-1BB may in fact, act as a costimulator ofB-cell activation. Future experiments will focus on characterization ofthe 4-1BB binding site(s) and determining if 4-1BB is involved in thymusdependent-B-cell proliferation and differentiation.

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[0441] The foregoing description has been directed to particularembodiments of the invention in accordance with the requirements of thePatent Statutes for the purposes of illustration and explanation. Itwill be apparent, however, to those skilled in this art that manymodifications and changes will be possible without departure from thescope and spirit of the invention. It is intended that the followingclaims be interpreted to embrace all such modifications.

1 13 1 2350 DNA Mus musculus misc_feature (1)...(2350) n = A,T,C or G 1atgtccatga actgctgagt ggataaacag cacgggatat ctctgtctaa aggaatatta 60ctacaccagg aaaaggacac attcgacaac aggaaaggag cctgtcacag aaaaccacag 120tgtcctgtgc atgtgacatt tcgccatggg aaacaactgt tacaacgtgg tggtcattgt 180gctgctgcta gtgggctgtg agaaggtggg agccgtgcag aactcctgtg ataactgtca 240gcctggtact ttctgcagaa aatacaatcc agtctgcaag agctgccctc caagtacctt 300ctccagcata ggtggacagc cgaactgtaa catctgcaga gtgtgtgcag gctatttcag 360gttcaagaag ttttgctcct ctacccacaa cgcggagtgt gagtgcattg aaggattcca 420ttgcttgggg ccacagtgca ccagatgtga aaaggactgc aggcctggcc aggagctaac 480gaagcagggt tgcaaaacct gtagcttggg aacatttaat gaccagaacg gtactggcgt 540ctgtcgaccc tggacgaact gctctctaga cggaaggtct gtgcttaaga ccgggaccac 600ggagaaggac gtggtgtgtg gaccccctgt ggtgagcttc tctcccagta ccaccatttc 660tgtgactcca gagggaggac caggagggca ctccttgcag gtccttacct tgttcctggc 720gctgacatcg gctttgctgc tggccctgat cttcattact ctcctgttct ctgtgctcaa 780atggatcagg aaaaaattcc cccacatatt caagcaacca tttaagaaga ccactggagc 840agctcaagag gaagatgctt gtagctgccg atgtccacag gaagaagaag gaggaggagg 900aggctatgag ctgtgatgta ctatcctagg agatgtgtgg gccgaaaccg agaagcacta 960ggaccccacc atcctgtgga acagcacaag caaccccacc accctgttct tacacatcat 1020cctagatgat gtgtgggcgc gcacctcatc caagtctctt ctaacgctaa catatttgtc 1080tttacctttt ttaaatcttt ttttaaattt aaattttatg tgtgtgagtg ttttgcctgc 1140ctgtatgcac acgtgtgtgt gtgtgtgtgt gtgacactcc tgatgcctga ggaggtcaga 1200agagaaaggg ttggttccat aagaactgga gttatggatg gctgtgagcc ggnnngatag 1260gtcgggacgg agacctgtct tcttatttta acgtgactgt ataataaaaa aaaaatgata 1320tttcgggaat tgtagagatt ctcctgacac ccttctagtt aatgatctaa gaggaattgt 1380tgatacgtag tatactgtat atgtgtatgt atatgtatat gtatatataa gactctttta 1440ctgtcaaagt caacctagag tgtctggtta ccaggtcaat tttattggac attttacgtc 1500acacacacac acacacacac acacacacgt ttatactacg tactgttatc ggtattctac 1560gtcatataat gggatagggt aaaaggaaac caaagagtga gtgatattat tgtggaggtg 1620acagactacc ccttctgggt acgtagggac agacctcctt cggactgtct aaaactcccc 1680ttagaagtct cgtcaagttc ccggacgaag aggacagagg agacacagtc cgaaaagtta 1740tttttccggc aaatcctttc cctgtttcgt gacactccac cccttgtgga cacttgagtg 1800tcatccttgc gccggaaggt caggtggtac ccgtctgtag gggcggggag acagagccgc 1860gggggagcta cgagaatcga ctcacagggc gccccgggct tcgcaaatga aactttttta 1920atctcacaag tttcgtccgg gctcggcgga cctatggcgt cgatccttat taccttatcc 1980tggcgccaag ataaaacaac caaaagcctt gactccggta ctaattctcc ctgccggccc 2040ccgtaagcat aacgcggcga tctccacttt aagaacctgg ccgcgttctg cctggtctcg 2100ctttcgtaaa cggttcttac aaaagtaatt agttcttgct ttcagcctcc aagcttctgc 2160tagtctatgg cagcatcaag gctggtattt gctacggctg accgctacgc cgccgcaata 2220agggtactgg gcggcccgtc gaaggccctt tggtttcaga aacccaaggc ccccctcata 2280ccaacgtttc gactttgatt cttgccggta cgtggtggtg ggtgccttag ctctttctcg 2340atagttagac 2350 2 256 PRT Mus musculus 2 Met Gly Asn Asn Cys Tyr Asn ValVal Val Ile Val Leu Leu Leu Val 1 5 10 15 Gly Cys Glu Lys Val Gly AlaVal Gln Asn Ser Cys Asp Asn Cys Gln 20 25 30 Pro Gly Thr Phe Cys Arg LysTyr Asn Pro Val Cys Lys Ser Cys Pro 35 40 45 Pro Ser Thr Phe Ser Ser IleGly Gly Gln Pro Asn Cys Asn Ile Cys 50 55 60 Arg Val Cys Ala Gly Tyr PheArg Phe Lys Lys Phe Cys Ser Ser Thr 65 70 75 80 His Asn Ala Glu Cys GluCys Ile Glu Gly Phe His Cys Leu Gly Pro 85 90 95 Gln Cys Thr Arg Cys GluLys Asp Cys Arg Pro Gly Gln Glu Leu Thr 100 105 110 Lys Gln Gly Cys LysThr Cys Ser Leu Gly Thr Phe Asn Asp Gln Asn 115 120 125 Gly Thr Gly ValCys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Arg 130 135 140 Ser Val LeuLys Thr Gly Thr Thr Glu Lys Asp Val Val Cys Gly Pro 145 150 155 160 ProVal Val Ser Phe Ser Pro Ser Thr Thr Ile Ser Val Thr Pro Glu 165 170 175Gly Gly Pro Gly Gly His Ser Leu Gln Val Leu Thr Leu Phe Leu Ala 180 185190 Leu Thr Ser Ala Leu Leu Leu Ala Leu Ile Phe Ile Thr Leu Leu Phe 195200 205 Ser Val Leu Lys Trp Ile Arg Lys Lys Phe Pro His Ile Phe Lys Gln210 215 220 Pro Phe Lys Lys Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala CysSer 225 230 235 240 Cys Arg Cys Pro Gln Glu Glu Glu Gly Gly Gly Gly GlyTyr Glu Leu 245 250 255 3 24 PRT Mus musculus 3 Cys Arg Val Cys Ala GlyTyr Phe Arg Phe Lys Lys Phe Cys Ser Ser 1 5 10 15 Thr His Asn Ala GluCys Glu Cys 20 4 22 PRT Drosophila 4 Cys Pro Val Cys Phe Asp Tyr Val IleLeu Gln Cys Ser Ser Gly His 1 5 10 15 Leu Val Cys Val Ser Cys 20 5 26PRT Dictyostelium 5 Cys Pro Ile Cys Phe Glu Phe Ile Tyr Lys Lys Gln IleTyr Gln Cys 1 5 10 15 Lys Ser Gly His His Ala Cys Lys Glu Cys 20 25 6 6PRT Mus musculus SITE (1)...(6) Xaa = Any Amino Acid 6 Val Gln Asn SerXaa Asp 1 5 7 12 PRT Artificial Sequence An artificial peptide 7 Cys ArgPro Gly Gln Glu Leu Thr Lys Ser Gly Tyr 1 5 10 8 24 PRT ArtificialSequence A conserved pattern 8 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Cys Xaa Xaa 1 5 10 15 Xaa His Xaa Xaa Xaa Cys Xaa Cys 20 9 4PRT Mus musculus 9 Cys Arg Cys Pro 1 10 4 PRT Artificial Sequence Aconsensus sequence 10 Cys Xaa Cys Pro 1 11 25 DNA Artificial Sequence Aprimer 11 acctcgaggt cctgtgcatg tgaca 25 12 25 DNA Artificial Sequence Aprimer 12 atgaattctt actgcaggag tgccc 25 13 11 PRT Mus musculus 13 CysArg Pro Gly Gln Glu Leu Thr Lys Gln Gly 1 5 10

I claim:
 1. A cDNA gene encoding for receptor protein 4-1BB.
 2. The cDNAgene of claim 1 having a nucleotide sequence as shown in FIGS. 2a and 2b.
 3. A cDNA gene of claim 1, identified as p4-1BB deposited at theAmerican Type Culture Collection at 12301 Parklawn Drive, Rockville, Md.20852 under ATCC No.
 67825. 4. The cDNA of claim 2 and fragments andderivatives thereof, wherein said fragments and derivatives can be usedas a probe to isolate DNA sequences encoding for proteins similar to thereceptor protein encoded by said cDNA.
 5. The cDNA of a human receptorcorresponding to the mouse cDNA 4-1BB isolated from a human source usingcDNA 4-1BB as a probe.
 6. The receptor protein 4-1BB produced by a)inserting the cDNA of 4-1BB into an appropriate expression vector, b)transfecting said expression vector into an appropriate transfectionhost, c) growing said transfected hosts in appropriate culture media andd) purifying the receptor protein from said culture media.
 7. A proteinhaving the amino acid sequence shown in FIGS. 2a and 2 b.
 8. The proteinof claim 7 and fragments and derivatives thereof, wherein said fragmentsand derivatives: a) can be used as a probe to isolate ligands toreceptor protein 4-1BB; b) can be used to stimulate proliferationB-cell's expressing 4-1BB ligands; or c) can be used to block 4-1BBligand binding.
 9. A monoclonal antibody against 4-1BB whichspecifically recognizes an epitope on the extracellular domain ofreceptor protein 4-1BB.
 10. The monoclonal antibody of claim 9 whereinsaid monoclonal antibody is produced from a hybridoma identified as 53A2and deposited at the American Type Culture Collection at 12301 ParklawnDrive, Rockville, Md. 20852 under ATCC No.: HB-11248.
 11. A hybridomacapable of producing a monoclonal antibody against 4-1BB whichspecifically recognizes an epitope on the extracellular domain ofreceptor protein 4-1BB.
 12. The hybridoma of claim 11 wherein saidhybridoma is identified as 53A2 and deposited at the American TypeCulture Collection at 12301 Parklawn Drive, Rockville, Md. 20852 underATCC No.: HB-11248.
 13. The method of using the monoclonal antibody ofclaim 9 to enhance T-cell proliferation comprising the step of treatingT-cells that have expressed receptor protein 4-1BB with said monoclonalantibody.
 14. The method of claim 13 further comprising the step ofconducting said treatment in the presence of protein tyrosinase kinase.15. The method of using the monoclonal antibody of claim 9 to enhanceT-cell activation comprising the step of treating T-cells that haveexpressed receptor protein 4-1BB with said monoclonal antibody.
 16. Themethod of claim 15 further comprising the step of conducting saidtreatment in the presence of protein tyrosinase kinase.
 17. A fusionprotein for detecting cell membrane ligands to receptor protein 4-1BB,comprising: a) at least a portion of said receptor protein 4-1BBcorresponding to the extracellular portion of said receptor protein4-1BB such that said portion of said receptor protein 4-1BB binds tosaid cell membrane ligands; and b) a detection protein bound to saidportion of said receptor protein 4-1BB such that ligand binding can bedetected by relative activity assays for said detection protein.
 18. Thefusion protein of claim 17 wherein said detection protein is alkalinephosphatase.
 19. A method of detecting cell membrane ligands to receptorprotein 4-1BB, comprising: a) providing a fusion protein including: 1)at least a portion of said receptor protein 4-1BB corresponding to theextracellular portion of said receptor protein 4-1BB such that saidportion of said receptor protein 4-1BB binds to said cell membraneligands, and 2) a detection protein bound to said portion of saidreceptor protein 4-1BB such that ligand binding can be detected byrelative activity assays for said detection protein; b) placing saidfusion protein in the presence of a cell suspected to express saidreceptor protein 4-1BB; c) washing said cell of any fusion protein notbound to said cell membrane ligands; d) placing said washed cells in thepresence of a substrate for said detection protein and measuring therelative activity of said detection protein.
 20. The method of claim 19wherein said detection protein is alkaline phosphatase.
 21. A method ofinducing B-cell proliferation comprising the step of treating B-cellsthat have expressed a ligand to receptor protein 4-1BB with cells thathave expressed receptor protein 4-1BB.